Synthetic chimeric poxviruses

ABSTRACT

The invention relates, in general, to synthetic chimeric poxviruses, compositions comprising such viruses, and the development and use of systems and methods for producing such synthetic chimeric poxviruses. The synthetic chimeric poxviruses are well suited for live virus vaccines and pharmaceutical formulations.

RELATED APPLICATIONS

This application claims priority and benefit from U.S. ProvisionalPatent Applications 62/434,794, filed Dec. 15, 2016, and 62/416,577,filed Nov. 2, 2016. The contents and disclosures of each of these priorprovisional applications are incorporated herein by reference in theirentirety.

A Sequence Listing associated with this application is being submittedelectronically via EFS-Web in text format, and is hereby incorporated byreference in its entirety into the specification. The name of the textfile containing the Sequence Listing is 104545-0026-101-SL.txt. The textfile, created on May 25, 2018, is 863,281 bytes in size.

BACKGROUND OF THE INVENTION

Poxviruses (members of the Poxviridae family) are double-stranded DNAviruses that can infect both humans and animals. Poxviruses are dividedinto two subfamilies based on host range. The Chordopoxviridaesubfamily, which infects vertebrate hosts, consists of eight genera, ofwhich four genera (Orthopoxvirus, Parapoxvirus, Molluscipoxvirus, andYatapoxvirus) are known to infect humans. Smallpox is caused byinfection with variola virus (VARV), a member of the genus Orthopoxvirus(OPV). The OPV genus comprises a number of genetically related andmorphologically identical viruses, including camelpox virus (CMLV),cowpox virus (CPXV), ectromelia virus (ECTV, “mousepox agent”), horsepoxvirus (HPXV), monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpoxvirus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus,vaccinia virus (VACV), variola virus (VARV) and volepox virus (VPV).Other than VARV, at least three other OPVs, including VACV, MPXV andCPXV, are known to infect humans. So far, vaccination with “live” VACVis the only proven protection against smallpox. An aggressive program ofvaccination led to the eradication of smallpox in 1980 and routinesmallpox vaccination of the public was stopped. However, a need remainsto find new safe and effective means of vaccinating individuals againstVARV and other OPVs.

A variety of preparations of VACV have been used as smallpox vaccines.Most of these comprised of a number of related viruses (e.g., Dryvax),and one comprises a single molecular clone, ACAM2000. However, likeDryvax and other VACV vaccines, even ACAM2000 is associated with seriousside effects including cardiomyopathy and pericarditis. To reduce risks,the ACAM2000 vaccine, like other live vaccines, has numerouscontraindications that preclude individuals with cancer,immunodeficiencies, organ transplant recipients, patients with atopicdermatitis, eczema, psoriasis, heart conditions, and patients onimmunosuppressants. It is estimated that 15-50% of the US populationwould fall under one of these categories, therefore confirming the needfor the development of a safer vaccine or vaccination protocol (Kennedyet al., 2007 Kennedy R, Poland G A. 2007. T-Cell epitope discovery forvariola and vaccinia viruses. Rev Med Viroll7: 93-113). Therefore, thereis a need for the development of a vaccine that is equivalent inefficacy to Dryvax or ACAM2000™, but that is safer.

The present invention provides chimeric poxviruses assembled andreplicated from chemically synthesized DNA. Because chemical genomesynthesis is not dependent on a natural template, a plethora ofstructural and functional modifications of the viral genome arepossible. Chemical genome synthesis is particularly useful when anatural template is not available for genetic replication ormodification by conventional molecular biology methods.

SUMMARY OF THE INVENTION

The present invention provides synthetic chimeric poxviruses (e.g.,synthetic chimeric OPV or scOPV), methods for producing such viruses andthe use of such viruses, for example, as immunogens, in immunogenicformulations, in in vitro assays, as vehicles for heterologous geneexpression, or as oncolytic agents. The synthetic chimeric poxviruses ofthe invention are characterized by one or more modifications relative toa wildtype poxvirus.

In part, the present invention relates to the discovery that a syntheticchimeric poxvirus (e.g., scOPV) can be produced from chemicallysynthesized overlapping fragments of the poxviral genome. Accordingly,the present invention, in part, provides synthetic chimeric poxviruses(e.g., scOPV) replicated and assembled from chemically synthesizednucleic acids. The disclosure also provides compositions comprising suchviruses. The disclosure further provides methods of using the poxvirusesproduced according to the methods of the disclosure.

In another aspect, the invention provides a method for protectingindividual humans and populations of humans against the consequences ofinfection with smallpox, pseudotypes of smallpox virus and other OPVsusing the synthetic chimeric poxviruses of the invention. In anotheraspect, the invention is a method for protecting individual humans andpopulations of humans against the consequences of infection withsmallpox (VARV) and pseudotypes of smallpox virus by using the syntheticchimeric poxviruses of the invention, with less toxicity, morbidity andmortality than available VACV-based vaccines. In certain aspects, theinvention provides a synthetic chimeric poxvirus (scPV) that isreplicated and reactivated from DNA derived from synthetic DNA, theviral genome of said virus differing from a wild type genome of saidvirus in that it is characterized by one or more modifications, themodifications being derived from a group comprising chemicallysynthesized DNA, cDNA or genomic DNA.

In some embodiments, the synthetic DNA is selected from one or more ofchemically synthesized DNA, PCR amplified DNA, engineered DNA andpolynucleotides comprising nucleoside analogs. In some embodiments, thesynthetic DNA is chemically synthesized DNA.

In some embodiments, the one or more modifications comprise one or moredeletions, insertions, substitutions, or a combination thereof. In someembodiments, the one or more modifications comprise one or moremodifications to introduce one or more unique restriction sites.

In some embodiments, the viral genome comprises heterologous terminalhairpin loops. In some embodiments, the viral genome comprises terminalhairpin loops derived from vaccinia virus. In some embodiments, the leftand right terminal hairpin loops a) comprise the slow form and the fastform of the vaccinia virus terminal hairpin loop, respectively, b)comprise the fast form and the slow form of the vaccinia virus terminalhairpin loop, respectively, c) both comprise the slow form of thevaccinia virus terminal hairpin loop, or d) both comprise the fast formof the vaccinia virus terminal loop.

In some embodiments, the virus is replicated and reactivated fromoverlapping chemically synthesized DNA fragments that correspond tosubstantially all of the viral genome of the scPV.

In some embodiments, the virus is replicated and reactivated from 1-14overlapping fragments. In some embodiments, the virus is replicated andreactivated from 8-12 overlapping fragments. In some embodiments, thevirus is replicated and reactivated from 10 overlapping fragments.

In some embodiments, the virus is reactivated using leporipoxvirus-catalyzed recombination and reactivation. In some embodiments, theleporipox virus is selected from the group consisting of: Shope fibromavirus (SFV), hare fibroma virus, rabbit fibroma virus, squirrel fibromavirus, and myxoma virus.

In certain aspects, the invention provides a synthetic chimeric orthopoxvirus (scOPV) that is replicated and reactivated from DNA derived fromsynthetic DNA, the viral genome of said virus differing from a wild typegenome of said virus in that it is characterized by one or moremodifications, the modifications being derived from a group comprisingchemically synthesized DNA, cDNA or genomic DNA.

In some embodiments, the synthetic DNA is selected from one or more of:chemically synthesized DNA, PCR amplified DNA, engineered DNA andpolynucleotides comprising nucleoside analogs. In some embodiments, thesynthetic DNA is chemically synthesized DNA.

In some embodiments, the OPV is selected from the group consisting of:camelpox (CMLV) virus, cowpox virus (CPXV), ectromelia virus (ECTV),horsepox virus (HPXV), monkeypox virus (MPXV), vaccinia virus (VACV),variola virus (VARV), rabbitpox virus (RPXV), raccoon poxvirus, skunkpoxvirus, Taterapox virus, Uasin Gishu disease virus, and volepox virus.

In some embodiments, the OPV is a VACV. In some embodiments, the viralgenome is based on the genome of VACV strain ACAM2000 and differs fromthe ACAM2000 genome in that it is characterized by one or moremodifications. In some embodiments, the viral genome is based on thegenome of VACV strain IOC and differs from the IOC genome in that it ischaracterized by one or more modifications. In some embodiments, theviral genome is based on the genome of VACV strain MVA and differs fromthe MVA genome in that it is characterized by one or more modifications.In some embodiments, the viral genome is based on the genome of VACVstrain MVA-BN and differs from the MVA-BN genome in that it ischaracterized by one or more modifications. In some embodiments, thewild type VACV genome is the genome of a strain selected from the groupconsisting of: Western Reserve, Clone 3, Tian Tian, Tian Tian clone TT9,Tian Tian clone TP3, NYCBH, Wyeth, Copenhagen, Lister 107, Lister-LO,IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR,Evans, Praha, LIVP, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, ConnaughtLaboratories, Serro 2, CM-01, Dryvax clone DPP13, Dryvax clone DPP15,Dryvax clone DPP20, Dryvax clone DPP17, Dryvax clone DPP21, andchorioallantois vaccinia virus Ankara.

In some embodiments, the one or more modifications comprise one or moredeletions, insertions, substitutions, or a combination thereof.

In some embodiments, the one or more modifications comprise one or moremodifications to introduce one or more unique restriction sites. In someembodiments, the one or more modifications comprise one or moremodifications to eliminate one or more restriction sites. In someembodiments, the one or more modifications comprise one or moremodifications to eliminate one or more AarI restriction sites. In someembodiments, the one or more modifications comprise one or moremodifications to eliminate all AarI restriction sites. In someembodiments, the one or more modifications comprise one or moremodifications to eliminate one more BsaI restriction sites.

In some embodiments, the viral genome comprises heterologous terminalhairpin loops. In some embodiments, the viral genome comprises terminalhairpin loops derived from vaccinia virus. In some embodiments, the leftand right terminal hairpin loops a) comprise the slow form and the fastform of the vaccinia virus terminal hairpin loop, respectively, b)comprise the fast form and the slow form of the vaccinia virus terminalhairpin loop, respectively, c) both comprise the slow form of thevaccinia virus terminal hairpin loop, or d) both comprise the fast formof the vaccinia virus terminal loop. In some embodiments, the slow formcomprises a nucleotide sequence that is at least 85% identical to thenucleotide sequence of SEQ ID NO: 11 and the fast form comprises anucleotide sequence that is at least 85% identical to the nucleotidesequence of SEQ ID NO: 12. In some embodiments, the slow form comprisesa nucleotide sequence that is at least 90% identical to the sequence ofSEQ ID NO: 11 and the fast form comprises a nucleotide sequence that isat least 90% identical to the nucleotide sequence of SEQ ID NO: 12. Insome embodiments, the slow form comprises a nucleotide sequence that isat least 95% identical to the nucleotide sequence of SEQ ID NO: 11 andthe fast form comprises a nucleotide sequence that is at least 95%identical to the nucleotide sequence of SEQ ID NO: 12. In someembodiments, the slow form consists of the nucleotide sequence of SEQ IDNO: 11 and the fast form consists of the nucleotide sequence of SEQ IDNO: 12.

In some embodiments, the virus is replicated and reactivated fromoverlapping chemically synthesized DNA fragments that correspond tosubstantially all of the viral genome of the OPV.

In some embodiments, the virus is replicated and reactivated from 1-14overlapping fragments. In some embodiments, the virus is replicated andreactivated from 8-12 overlapping fragments. In some embodiments, thevirus is replicated and reactivated from 10 overlapping fragments.

In some embodiments, the virus is reactivated using leporipoxvirus-catalyzed recombination and reactivation. In some embodiments, theleporipox virus is selected from the group consisting of: Shope fibromavirus (SFV), hare fibroma virus, rabbit fibroma virus, squirrel fibromavirus, and myxoma virus.

In certain aspects, the invention provides a synthetic chimeric horsepoxvirus (scHPXV) that is replicated and reactivated from synthetic DNA,the viral genome differing from a wild type genome of HPXV in that it ischaracterized by one or more modifications, the modifications beingderived from a group comprising chemically synthesized DNA, cDNA orgenomic DNA.

In some embodiments, the synthetic DNA is selected from one or more of:chemically synthesized DNA, PCR amplified DNA, engineered DNA andpolynucleotides comprising nucleoside analogs. In some embodiments, thesynthetic DNA is chemically synthesized DNA.

In some embodiments, the viral genome is based on the genome of HPXVstrain MNR-76 and differs from the MNR-76 genome in that it ischaracterized by one or more modifications. In some embodiments, the oneor more modifications comprise one or more deletions, insertions,substitutions, or a combination thereof.

In some embodiments, the one or more modifications comprise one or moremodifications to introduce one or more unique restriction sites. In someembodiments, the one or more modifications are present in HPXV044 orHPXV095. In some embodiments, the one or more modifications comprise oneor more mutations listed in Table 3. In some embodiments, the one ormore modifications comprise one or more modifications to eliminate oneor more restriction sites. In some embodiments, the one or moremodifications comprise one or more modifications to eliminate one ormore AarI restriction sites. In some embodiments, the one or moremodifications comprise one or more modifications to eliminate all AarIrestriction sites. In some embodiments, the one or more modificationscomprise one or more modifications to eliminate one or more BsaIrestriction sites. In some embodiments, the one or more modificationscomprise one or more mutations listed in Table 2.

In some embodiments, the viral genome comprises heterologous terminalhairpin loops. In some embodiments, the viral genome comprises terminalhairpin loops derived from vaccinia virus. In some embodiments, the leftand right terminal hairpin loops a) comprise the slow form and the fastform of the vaccinia virus terminal hairpin loop, respectively, b)comprise the fast form and the slow form of the vaccinia virus terminalhairpin loop, respectively, c) both comprise the slow form of thevaccinia virus terminal hairpin loop, or d) both comprise the fast formof the vaccinia virus terminal loop. In some embodiments, the slow formcomprises a nucleotide sequence that is at least 85% identical to thesequence of SEQ ID NO: 11 and the fast form comprises a nucleotidesequence that is at least 85% identical to the nucleotide sequence ofSEQ ID NO: 12. In some embodiments, the slow form comprises a nucleotidesequence that is at least 90% identical to the sequence of SEQ ID NO: 11and the fast form comprises a nucleotide sequence that is at least 90%identical to the nucleotide sequence of SEQ ID NO: 12. In someembodiments, the slow form comprises a nucleotide sequence that is atleast 95% identical to the sequence of SEQ ID NO: 11 and the fast formcomprises a nucleotide sequence that is at least 95% identical to thenucleotide sequence of SEQ ID NO: 12. In some embodiments, the slow formconsists of the nucleotide sequence of SEQ ID NO: 11 and the fast formconsists of the nucleotide sequence of SEQ ID NO: 12. In someembodiments, the viral genome comprises terminal hairpin loops derivedfrom camelpox virus, cowpox virus, ectromelia virus, monkeypox virus,variola virus, rabbitpox virus, raccoonpox virus, skunkpox virus,Taterapox virus, Uasin Gishu disease virus, or volepox virus.

In some embodiments, the virus is replicated and assembled fromoverlapping chemically synthesized DNA fragments that correspond tosubstantially all of the viral genome of HPXV.

In some embodiments, the virus is replicated and reactivated from 1-14overlapping fragments. In some embodiments, the virus is replicated andreactivated from 8-12 overlapping fragments. In some embodiments, thevirus is replicated and reactivated from 10 overlapping fragments.

In some embodiments, the virus is reactivated using leporipoxvirus-catalyzed recombination and reactivation. In some embodiments, theleporipox virus is selected from the group consisting of: Shope fibromavirus (SFV), hare fibroma virus, rabbit fibroma virus, squirrel fibromavirus, and myxoma virus.

In certain aspects, the invention provides a method of producing asynthetic chimeric poxvirus (scPV) comprising the steps of: (i)chemically synthesizing overlapping DNA fragments that correspond tosubstantially all of the viral genome of the poxvirus; (ii) transfectingthe overlapping DNA fragments into helper virus-infected cells; (iii)culturing said cells to produce a mixture of helper virus and syntheticchimeric poxviral particles in said cells; and (iv) plating the mixtureon host cells specific to the scPV to recover the scPV.

In some embodiments, the helper virus is a leporipox virus. In someembodiments, the leporipox virus is selected from the group consistingof: Shope fibroma virus (SFV), hare fibroma virus, rabbit fibroma virus,squirrel fibroma virus, and myxoma virus. In some embodiments, theleporipox virus is SFV.

In some embodiments, the helper virus is fowlpox virus.

In some embodiments, the helper virus is a psoralen-inactivated helpervirus.

In some embodiments, the helper virus-infected cells are BGMK cells.

In some embodiments, step (i) further comprises chemically synthesizingterminal hairpin loops from a poxvirus and ligating them onto thefragments comprising the left and right termini of the viral genome.

In certain aspects, the invention provides a method of producing asynthetic chimeric orthopox virus (scOPV) comprising the steps of: (i)chemically synthesizing overlapping DNA fragments that correspond tosubstantially all of the viral genome of the OPV; (ii) transfecting theoverlapping DNA fragments into helper virus-infected cells; (iii)culturing said cells to produce a mixture of helper virus and scOPVparticles in said cells; and (iv) plating the mixture on OPV-specifichost cells to recover the scOPV.

In some embodiments, the helper virus is a leporipox virus. In someembodiments, the leporipox virus is selected from the group consistingof: Shope fibroma virus (SFV), hare fibroma virus, rabbit fibroma virus,squirrel fibroma virus, and myxoma virus. In some embodiments, theleporipox virus is SFV.

In some embodiments, the helper virus is fowlpox virus.

In some embodiments, the helper virus is a psoralen-inactivated helpervirus.

In some embodiments, the helper virus-infected cells are BGMK cells.

In some embodiments, the OPV-specific host cells are BSC-40 cells.

In some embodiments, the OPV is selected from the group consisting of:camelpox virus, cowpox virus, ectromelia virus, horsepox virus,monkeypox virus, vaccinia virus, variola virus, rabbitpox virus, raccoonpoxvirus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus,volepox virus.

In some embodiments, step (i) further comprises chemically synthesizingterminal hairpin loops from an OPV and ligating them onto the fragmentscomprising the left and right termini of the viral genome.

In certain aspects, the invention provides a method of producing asynthetic chimeric horsepox virus (scHPXV) comprising the steps of: (i)chemically synthesizing overlapping DNA fragments that correspond tosubstantially all of the HPXV genome; (ii) transfecting the overlappingDNA fragments into helper virus-infected cells; (iii) culturing saidcells to produce a mixture of helper virus and scHPXV particles in saidcells; and (iv) plating the mixture on HPXV-specific host cells torecover the scHPXV.

In certain aspects, the invention provides a method of producing asynthetic chimeric horsepox virus (scHPXV) comprising: (i) chemicallysynthesizing overlapping DNA fragments that correspond to substantiallyall of the HPXV genome; (ii) transfecting the overlapping DNA fragmentsinto Shope fibroma virus (SFV)-infected cells; (iii) culturing saidcells to produce a mixture of SFV and scHPXV particles in said cells;and (iv) plating the mixture on HPXV-specific host cells to recover thescHPXV.

In some embodiments, the helper virus is a leporipox virus. In someembodiments, the leporipox virus is selected from the group consistingof: Shope fibroma virus (SFV), hare fibroma virus, rabbit fibroma virus,squirrel fibroma virus, and myxoma virus. In some embodiments, theleporipox virus is SFV.

In some embodiments, the helper virus is fowlpox virus.

In some embodiments, the helper virus is a psoralen-inactivated helpervirus.

In some embodiments, the helper virus-infected cells are BGMK cells.

In some embodiments, the HPXV-specific host cells are BSC-40 cells.

In some embodiments, step (i) further comprises chemically synthesizingterminal hairpin loops from an OPV and ligating them onto the fragmentscomprising the left and right termini of the HPXV genome.

In some embodiments, the overlapping DNA fragments comprise: i)nucleotide sequences that are at least 85% identical to the sequences ofSEQ ID NOs: 1-10; ii) nucleotide sequences that are at least 90%identical to the sequences of SEQ ID NOs: 1-10; (iii) nucleotidesequences that are at least 95% identical to the sequences of SEQ IDNOs: 1-10; or (iv) nucleotide sequences that consist of the sequences ofSEQ ID NOs: 1-10.

In some embodiments, the SFV-infected cells are BGMK cells.

In some embodiments, the HPXV-specific host cells are BSC-40 cells.

In certain aspects, the invention provides a synthetic chimeric poxvirus(scPV) generated by the methods of the disclosure.

In certain aspects, the invention provides a synthetic chimeric orthopoxvirus (scOPV) generated by the methods of the disclosure.

In certain aspects, the invention provides a synthetic chimeric horsepoxvirus (scHPXV) generated by methods of the disclosure.

In certain aspects, the invention provides compositions comprising apharmaceutically acceptable carrier and an scPV of the disclosure.

In certain aspects, the invention provides compositions comprising apharmaceutically acceptable carrier and an scOPV of the disclosure.

In certain aspects, the invention provides a method of triggering orboosting an immune response against variola virus, comprisingadministering to a subject in need thereof a composition comprising anscOPV of the disclosure.

In certain aspects, the invention provides a method of triggering orboosting an immune response against vaccinia virus, comprisingadministering to a subject in need thereof a composition comprising anscOPV of the disclosure.

In certain aspects, the invention provides a method of triggering orboosting an immune response against monkeypox virus, comprisingadministering to a subject in need thereof a composition comprising anscOPV of the disclosure.

In certain aspects, the invention provides a method of immunizing ahuman subject to protect said subject from variola virus infection,comprising administering to said subject a composition comprising anscOPV of the disclosure.

In certain aspects, the invention provides a method of treating avariola virus infection, comprising administering to a subject in needthereof a composition comprising an scOPV of the disclosure.

In certain aspects, the invention provides a composition comprising apharmaceutically acceptable carrier and an scHPXV of the disclosure.

In certain aspects, the invention provides a method of triggering orboosting an immune response against variola virus, comprisingadministering to a subject in need thereof a composition comprising anscHPXV of the disclosure.

In certain aspects, the invention provides a method of triggering orboosting an immune response against vaccinia virus, comprisingadministering to a subject in need thereof a composition comprising anscHPXV of the disclosure.

In certain aspects, the invention provides a method of triggering orboosting an immune response against monkeypox virus, comprisingadministering to a subject in need thereof a composition comprising anscHPXV of the disclosure.

In certain aspects, the invention provides a method of immunizing ahuman subject to protect said subject from variola virus infection,comprising administering to said subject a composition comprising anscHPXV of the disclosure.

In certain aspects, the invention provides a method of treating avariola virus infection, comprising administering to a subject in needthereof a composition comprising an scHPXV of the disclosure.

In certain aspects, the invention provides a kit comprising acomposition comprising an scPV of the disclosure.

In certain aspects, the invention provides a kit comprising acomposition comprising an OPV of the disclosure.

In certain aspects, the invention provides a kit comprising acomposition comprising the scHPXV of the disclosure.

In certain aspects, a composition of the invention is administered in apoxvirus treatment facility. In certain aspects, a poxvirus treatmentfacility is a facility wherein subjects in need of immunization ortreatment with a composition or method of the invention may be immunizedor treated in an environment such that they are sequestered from othersubjects not intended to be immunized or treated or who might bepotentially infected by the treated subject (e.g., caregivers andhousehold members). In some embodiments, the subjects not intended to beimmunized or potentially infected by the treated subject, include HIVpatients, patients undergoing chemotherapy, patients undergoingtreatment for cancer, rheumatologic disorders, or autoimmune disorders,patients who are undergoing or have received an organ or tissuetransplant, patients with immune deficiencies, children, pregnant women,patients with atopic dermatitis, eczema, psoriasis, heart conditions,and patients on immunosuppressants, etc. In some embodiments, thepoxvirus treatment facility is an orthopoxvirus treatment facility. Insome embodiments, the poxvirus treatment facility is a smallpoxtreatment facility.

In certain aspects, a composition of the invention is administered by aspecialist in smallpox adverse events. In some embodiments, the smallpoxadverse events include but are not limited to eczema vaccinatum,progressive vaccinia, postvaccinal encephalitis, myocarditis, anddilated cardiomyopathy.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application contains at least one drawing executed in color.Copies of this patent application with color drawings will be providedby the Office upon request and payment of the necessary fee.

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the inventionthere are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

FIGS. 1A and 1B. Schematic representation of the linear dsDNA HPXVgenome (strain MNR; Genbank Accession DQ792504). A. FIG. 1A illustratesthe unmodified genome sequence of HPXV genome with individual HPXV genes(purple) and the naturally occurring AarI and BsaI sites indicated. B.FIG. 1B depicts the modified synthetic chimeric HPXV (scHPXV) genomethat was chemically synthesized using the overlapping genomic DNAfragments (shown in red). The engineered SapI restriction sites thatwere used to ligate the VACV terminal hairpin loops onto the ITRs, alongwith the unmodified BsaI sites in the left and right ITR fragments, arealso shown. The SapI sites were located in plasmid vector sitesimmediately to the left and right ends of the Left Inverted TerminalRepeat (LITR) and Right Inverted Terminal Repeat (RITR), respectively.

FIG. 2(A-C). Detailed schematic representation of the modified scHPXVYFP-gpt::095 genome and VACV (WR strain) terminal hairpin loops. A. FIG.2A depicts the modified scHPXV YFP-gpt::095 genome. The unmodified BsaIsites are shown as blue lines on the genome. The novel AvaI and StuIrestriction sites that were created in HPXV044 (the VACV F4L homolog)are also marked (green lines). The location of the selectable markeryellow fluorescent protein/guanosine phosphoribosyl transferase(yfp/gpt) in the HPXV095 locus (the VACV J2R homolog) of Frag_3 is alsoshown (yellow). B. FIG. 2B depicts the nucleotide sequence of the S (SEQID NO: 11) and F (SEQ ID NO: 12) forms of the terminal hairpin loop, andthe color coding is explained in (C). C. FIG. 2C depicts the secondarystructure predictions of the F and S forms of terminal hairpin loopsthat are covalently attached to the terminal ends of the linear dsDNAgenomes of VACV. The terminal loop sequence is highlighted in green. Theconcatamer resolution sequence is boxed in red.

FIGS. 3A and 3B. The ˜70 bp VACV terminal hairpin can be ligated to theleft and right HPXV ITR fragments. A. FIG. 3A depicts a schematicdiagram of the left and right HPXV ITR fragments marking the locationsof the SapI and PvuII recognition sites. The predicted fragment sizes ofthe DNA following digestion with SapI and PvuII are shown. B. FIG. 3Bdepicts agarose gel electrophoresis of the left and right ITR fragmentsfollowing ligation of the ˜70 bp terminal hairpin to the 1472 bp ITRfragment cut with SapI. The ligated DNAs were subsequently cut withPvuII to facilitate detection of the small change in size caused by theaddition of the hairpins.

FIGS. 4A-4C. PCR analysis and restriction digestion of scHPXVYFP-gpt::095 genomes confirm successful reactivation of scHPXVYFP-gpt::095. A. FIG. 4A depicts the results of PCR analysis of scHPXVYFP-gpt::095 clones. Primers that flank conserved BsaI restriction sitesin both VACV and scHPXV YFP-gpt::095 were used to amplify a series of ˜1kbp products. The PCR products were subsequently digested with BsaI andthe resulting DNA fragments were separated by agarose gelelectrophoresis. VACV is cut, but all the BsaI sites have been deletedfrom two different scHPXV YFP-gpt::095 clones. B. FIG. 4B depicts pulsefield gel electrophoresis (PFGE) of VACV-WR and scHPXV YFP-gpt::095genomic DNAs. Virus DNAs was digested with BsaI, HindIII, or leftuntreated, and were then separated on a 1% Seakem gold agarose gel for14 h at 14° C. at 5.7V/cm with a switch time of 1 to 10 seconds. Aslight difference in size between the intact VACV and scHPXVYFP-gpt::095 genomes was observed. The faint bands marked with anasterisk (*) are either incomplete DNA digestion products or could becut mitochondrial DNA fragments that often contaminate VACV virionpreparations. C. FIG. 4C depicts conventional agarose gelelectrophoresis of VACV-WR and scHPXV YFP-gpt::095 genomic DNA digestedwith BsaI or HindIII. DNA fragments were visualized by staining gelswith SybrGold DNA stain.

FIGS. 5A and 5B. VACV terminal hairpins were successfully ligated ontothe scHPXV YFP-gpt::095 ITR fragments. A. Illumina sequence reads weremapped to the scHPXV YFP-gpt::095 reference sequence. The SapI site (inthe vector, top strand sequence) was cut to create the point forligating the telomeric hairpins onto the left and right ITR fragments.The location of the terminal loop of the VACV-derived terminal hairpinis shown, along with the concatamer resolution site. The first A in theconcatamer resolution site is also the first nucleotide reported forHBXV (NCBI DQ792504), the remainder of the sequence to the left of that“A” was designed using VACV as a reference. Note that the “C's” weresimply added as a scaffold to avoid the truncation of the reads by theassembly program. B. A subset comprising the longest Illumina sequencingreads, extending beyond the known HPXV sequence, are shown alignedagainst a polydC template. These reads span the entire length of anunfolded hairpin as was originally provided using syntheticoligonucleotides. Because of the inverted terminal duplications inpoxviruses, all of the reads from the left and right ends “pile up”together and any original orientation or distribution is lost. It isapparent that both F- and S-forms of the VACV hairpin are detected andthe ratio of F- to S-reads in this region was 1.03±0.01 (SEM) in eightdifferent virus-sequencing reactions.

FIGS. 6A-6C. The BsaI sites in scHPXV YFP-gpt::095 region 96,050 to96,500 were correctly mutated. A. FIG. 6A depicts Illumina sequencereads that mapped to one region of the HPXV (DQ792504) genome. Only asmall fraction of the reads are shown. The conflicts in the sequencingreads at pos. 96,239 and pos. 96437 are highlighted in blue and yellow,respectively. B. FIG. 6B depicts the magnification of the Illuminasequencing reads from scHPXV YFP-gpt::095 that mapped to HPXV (DQ792504)near pos. 96239. A nucleotide substitution (T96239C) was detected (referto Table 2). C. FIG. 6C depicts the magnification of the Illuminasequencing reads from scHPXV YFP-gpt::095 that mapped to HPXV (DQ792504)at pos. 96437. A nucleotide substitution (A96437C) was detected (referto Table 2). These mutations were introduced into the clones used toassemble scHPXV YFP-gpt::095 so as to delete undesirable BsaIrecognition sites (GGTCTC).

FIGS. 7A-7C. Nucleotide changes in the HPXV044 gene of scHPXVYFP-gpt::095 that were used to create the unique restriction sites AvaIand StuI. A. FIG. 7A depicts Illumina sequence reads that mapped to theHPXV044 gene in the HPXV (DQ792504) genome. Only a small fraction of thereads are shown. HPXV044 encodes the HPXV homolog of VACV F4L. The siteswhere the sequence reads do not match to the original HPXV (DQ792504)sequence are highlighted in yellow vertical lines. B. FIG. 7B depictsthe magnification of the Illumina sequencing reads from scHPXVYFP-gpt::095 that mapped to HPXV (DQ792504) at pos. 44,512. A nucleotidesubstitution (T44512G) was identified which creates an AvaI site(CYCGRG; refer to Table 2). C. FIG. 7C depicts the magnification of theIllumina sequencing reads from scHPXV YFP-gpt::095 that mapped to HPXV(DQ792504) at pos. 45,061. A nucleotide substitution (T45061G) wasdetected that creates a StuI site (AGGCCT; refer to Table 2).

FIGS. 8A-8C. ScHPXV YFP-gpt::095 grows like other Orthopoxviruses butexhibits a small plaque phenotype in BSC-40 cells. A. FIG. 8Aillustrates the multi-step growth of VACV-WR, DPP15, CPXV, and scHPXVYFP-gpt::095 in BSC-40 (top left panel), HeLa (top middle panel),primary HEL (top right panel), and Vero (bottom left panel) cell lines.B. FIG. 8B illustrates plaque size comparisons between VACV-WR, DPP15,CPXV, and scHPXV YFP-gpt::095. BSC-40 cells were infected with theindicated viruses and at 48 h post infection the cells were fixed andstained. The areas (in arbitrary units [A.U.]) of 24 plaques over threeindependent experiments were measured for each virus. Data are expressedas the mean plaque diameter. **, P<0.01; ****, P<0.0001. C. FIG. 8Cdepicts plaque morphology of BSC-40 cells infected with the indicatedviruses for 72 h. Cells were fixed, stained, and scanned forvisualization.

FIG. 9. Schematic representation of the linear dsDNA genome of VACV(strain ACAM2000; Genbank Accession AY313847). The unmodified genomesequence of VACV ACAM2000 is illustrated with naturally occurring AarIand BsaI recognition sites marked. The overlapping DNA fragments aredepicted in blue. The left (LITR_ACAM2000) and right (RITR_ACAM2000)fragments are shown in orange.

FIG. 10. A graphical representation of the % weight loss over time afteradministration of various compositions and doses to mice. The depicteddata are generated from groups of 5 female BALB/c mice that areinoculated with the indicated dose of scHPXV YFP-gpt::095 (alsodesignated as scHPXV (ΔHPXV_095/J2R) or scHPXV (yfp/gpt)), scHPXV (wt),Dryvax DPP15, or VACV WR in 10 μl of PBS. Mice are weighed daily for 28days and any that lost>25% of their initial weight are euthanized. Datapoints represent mean scores, and error bars represent standarddeviation.

FIGS. 11A and 11B. Graphical representations of the % weight loss overtime after administration of various compositions and doses to mice. Thedepicted data are generated from mice that are previously vaccinated(FIG. 10) and who are then challenged with a lethal dose of VACV WR (10⁶PFU) intranasally. FIG. 11A shows the weight changes and FIG. 11B showsthe clinical scores in mice recorded daily for 13 days. Any mice thatlost>25% of their initial weight are euthanized. Mice are assigned aclinical score based upon the appearance of ruffled fur, hunchedposture, difficulty breathing, and decreased mobility. Data pointsrepresent mean differences in weights or scores, and error barsrepresent standard deviation. † indicate the number of mice that succumbto the VACV infection on a given day.

FIG. 12. Graphical representation of the % survival over time afteradministration of various compositions and doses to mice. The depicteddata are generated from mice that are previously vaccinated (FIG. 10)and who are then challenged with a lethal dose of VACV WR (10⁶ PFU)intranasally. FIG. 12 shows survival curves of mice who are challengedintranasally with a lethal dose of VACV WR (10⁶ PFU). † indicate thenumber of mice that succumb to the VACV infection on the indicated day.

FIGS. 13A and 13B. Characterization of VACV-HPXV hybrid viruses. A. HPXVinserts in VACV strain WR. Virus genomes were sequenced using anIllumina platform, assembled, and LAGAN³² and “Base-by-Base”³³ softwarewere used to align and generate the maps shown. Places where VACVsequences (white) have been replaced by HPXV sequences are color codedaccording to the difference. The first hybrid virus (“VACV/HPXV+fragment3”) was obtained by co-transfecting VACV DNA with HPXV Fragment_3 intoSFV-infected cells. The green-tagged insertion encodes the YFP-gptselection marker. Clones 1-3 were obtained by purifying the DNA fromthis first hybrid genome and transfecting it again, along with HPXVfragments 2, 4, 5, and 7, into SFV-infected cells. B. A PCR-basedscreening approach for identifying hybrid and reactivated viruses. PCRprimers designed to target both HPXV and VACV and used to amplify DNAsegments spanning the BsaI sites that were mutated in the synthetic HPXVclones. Following PCR amplification, the products were digested withBsaI to differentiate VACV sequences (which cut) from HPXV (which do notcut). The VACV/HPXV hybrids exhibit a mix of BsaI sensitive andresistant sites whereas the reactivated scHPXV YFP-gpt::095 clone isfully BsaI resistant.

FIGS. 14A-14C. Growth properties of scHPXV versus scHPXV YFP-gpt::095.A. Plaque size measurements. Homologous recombination was used toreplace the YFP-gpt locus in scHPXV YFP-gpt::095 with thymidine kinasegene sequences. This produced a virus with a fully wild-type complementof HPXV genes (scHPXV). BSC-40 cells were infected with the indicatedviruses and cultured for three days. The dishes were stained and theplaque areas measured using a scanned digital image. Statisticallysignificant differences are noted ****P<0.0001). B. Plaque images. C.Multi-step virus growth in culture. The indicated cell lines wereinfected with scHPXV or scHPXV YFP-gpt::095 at a multiplicity ofinfection of 0.01, the virus harvested at the indicated times, andtitrated on BSC-40 cells in triplicate. No significant differences inthe growth of these viruses were detected in these in vitro assays.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, pharmacology, cell and tissueculture, molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, genetics and proteinand nucleic acid chemistry, described herein, are those well-known andcommonly used in the art. In case of conflict, the presentspecification, including definitions, will control.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Gene Transfer Vectors for Mammalian Cells (J. M. Millerand M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: ALaboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001); Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, N Y (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); Coligan et al., Short Protocols inProtein Science, John Wiley & Sons, N Y (2003); Short Protocols inMolecular Biology (Wiley and Sons, 1999).

Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry,biochemistry, immunology, molecular biology, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, and chemical analyses.

Throughout this specification and embodiments, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

Any example(s) following the term “e.g.” or “for example” is not meantto be exhaustive or limiting.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

The articles “a”, “an” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. Reference to “about” a value or parameter herein includes(and describes) embodiments that are directed to that value or parameterper se. For example, description referring to “about X” includesdescription of “X.” Numeric ranges are inclusive of the numbers definingthe range.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g., 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,and also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the embodimented invention.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Definitions

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the terms “wild type virus”, “wild type genome”, “wildtype protein,” or “wild type nucleic acid” refer to a sequence of aminoor nucleic acids that occurs naturally within a certain population(e.g., a particular viral species, etc.).

The terms “chimeric” or “engineered” or “modified” (e.g., chimericpoxvirus, engineered polypeptide, modified polypeptide, engineerednucleic acid, modified nucleic acid) or grammatical variations thereofare used interchangeably herein to refer to a non-native sequence thathas been manipulated to have one or more changes relative a nativesequence.

As used herein, “synthetic virus” refers to a virus initially derivedfrom synthetic DNA (e.g., chemically synthesized DNA, PCR amplified DNA,engineered DNA, polynucleotides comprising nucleoside analogs, etc., orcombinations thereof) and includes its progeny, and the progeny may notnecessarily be completely identical (in morphology or in genomic DNAcomplement) to the original parent synthetic virus due to natural,accidental, or deliberate mutation. In some embodiments, the syntheticvirus refers to a virus where substantially all of the viral genome isinitially derived from chemically synthesized DNA.

As outlined elsewhere herein, certain positions of the viral genome canbe altered. By “position” as used herein is meant a location in thegenome sequence. Corresponding positions are generally determinedthrough alignment with other parent sequences.

As used herein, “residue” refers to a position in a protein and itsassociated amino acid identity.

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog;internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.); those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.); those with intercalators (e.g.,acridine, psoralen, etc.); those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.); those containingalkylators; those with modified linkages (e.g., alpha anomeric nucleicacids, etc.); as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. The chain may be linear or branched, it may comprise modifiedamino acids, and/or may be interrupted by non-amino acids. The termsalso encompass an amino acid chain that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that the polypeptides can occur as single chains orassociated chains.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalwith the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

As used herein, a “host cell” includes an individual cell or cellculture that can be or has been a recipient for vector(s) forincorporation of polynucleotide inserts. Host cells include progeny of asingle host cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation. A hostcell includes cells transfected and/or transformed in vivo with anucleic acid of this invention.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “isolated molecule” (where the molecule is, for example,a polypeptide, a polynucleotide, or fragment thereof) is a molecule thatby virtue of its origin or source of derivation (1) is not associatedwith one or more naturally associated components that accompany it inits native state, (2) is substantially free of one or more othermolecules from the same species (3) is expressed by a cell from adifferent species, or (4) does not occur in nature. Thus, a moleculethat is chemically synthesized, or expressed in a cellular systemdifferent from the cell from which it naturally originates, will be“isolated” from its naturally associated components. A molecule also maybe rendered substantially free of naturally associated components byisolation, using purification techniques well known in the art. Moleculepurity or homogeneity may be assayed by a number of means well known inthe art. For example, the purity of a polypeptide sample may be assayedusing polyacrylamide gel electrophoresis and staining of the gel tovisualize the polypeptide using techniques well known in the art. Forcertain purposes, higher resolution may be provided by using HPLC orother means well known in the art for purification.

As used herein, the term “isolated”, in the context of viruses, refersto a virus that is derived from a single parental virus. A virus can beisolated using routine methods known to one of skill in the artincluding, but not limited to, those based on plaque purification andlimiting dilution.

As used herein, the phrase “multiplicity of infection” or “MOI” is theaverage number of viruses per infected cell. The MOI is determined bydividing the number of virus added (ml added×plaque forming units (PFU))by the number of cells added (ml added×cells/ml).

As used herein, “purify,” and grammatical variations thereof, refers tothe removal, whether completely or partially, of at least one impurityfrom a mixture containing the polypeptide and one or more impurities,which thereby improves the level of purity of the polypeptide in thecomposition (i.e., by decreasing the amount (ppm) of impurity(ies) inthe composition). As used herein “purified” in the context of virusesrefers to a virus which is substantially free of cellular material andculture media from the cell or tissue source from which the virus isderived. The language “substantially free of cellular material” includespreparations of virus in which the virus is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, virus that is substantially free of cellular materialincludes preparations of protein having less than about 30%, 20%, 10%,or 5% (by dry weight) of cellular protein (also referred to herein as a“contaminating protein”). The virus is also substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the virus preparation. A virus can bepurified using routine methods known to one of skill in the artincluding, but not limited to, chromatography and centrifugation.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

The terms “patient”, “subject”, or “individual” are used interchangeablyherein and refer to either a human or a non-human animal. These termsinclude mammals, such as humans, primates, livestock animals (includingbovines, porcines, camels, etc.), companion animals (e.g., canines,felines, etc.) and rodents (e.g., mice and rats).

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of, or a reduction in oneor more symptoms of a disease (e.g., a poxviral infection) in a subjectas a result of the administration of a therapy (e.g., a prophylactic ortherapeutic agent). For example, in the context of the administration ofa therapy to a subject for an infection, “prevent”, “preventing” and“prevention” refer to the inhibition or a reduction in the developmentor onset of an infection (e.g., a poxviral infection or a conditionassociated therewith), or the prevention of the recurrence, onset, ordevelopment of one or more symptoms of an infection (e.g., a poxviralinfection or a condition associated therewith), in a subject resultingfrom the administration of a therapy (e.g., a prophylactic ortherapeutic agent), or the administration of a combination of therapies(e.g., a combination of prophylactic or therapeutic agents).

“Treating” a condition or patient refers to taking steps to obtainbeneficial or desired results, including clinical results. With respectto infections (e.g., a poxviral infection), treatment refers to theeradication or control of the replication of an infectious agent (e.g.,a poxvirus), the reduction in the numbers of an infectious agent (e.g.,the reduction in the titer of poxvirus), the reduction or ameliorationof the progression, severity, and/or duration of an infection (e.g., apoxviral infection or a condition or symptoms associated therewith), orthe amelioration of one or more symptoms resulting from theadministration of one or more therapies (including, but not limited to,the administration of one or more prophylactic or therapeutic agents).With respect to cancer, treatment refers to the eradication, removal,modification, or control of primary, regional, or metastatic cancertissue that results from the administration of one or more therapeuticagents of the invention. In certain embodiments, such terms refer to theminimizing or delaying the spread of cancer resulting from theadministration of one or more therapeutic agents of the invention to asubject with such a disease. In other embodiments, such terms refer toelimination of disease-causing cells.

“Administering” or “administration of” a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered sublingually or intranasally, by inhalation into thelung or rectally. Administering can also be performed, for example,once, a plurality of times, and/or over one or more extended periods. Insome aspects, the administration includes both direct administration,including self-administration, and indirect administration, includingthe act of prescribing a drug. For example, as used herein, a physicianwho instructs a patient to self-administer a drug, or to have the drugadministered by another and/or who provides a patient with aprescription for a drug is administering the drug to the patient.

Each embodiment described herein may be used individually or incombination with any other embodiment described herein.

Overview

Poxviruses are large (˜200 kbp) DNA viruses that replicate in thecytoplasm of infected cells. The Orthopoxvirus (OPV) genus comprises anumber of poxviruses that vary greatly in their ability to infectdifferent hosts. Vaccinia virus (VACV), for example, can infect a broadgroup of hosts, whereas variola virus (VARV), the causative agent ofsmallpox, only infects humans. A feature common to many, if not allpoxviruses, is their ability to non-genetically “reactivate” within ahost. Non-genetic reactivation refers to a process wherein cellsinfected by one poxvirus can promote the recovery of a second “dead”virus (for example one inactivated by heat) that would be non-infectiouson its own.

Purified poxvirus DNA is not infectious because the virus life cyclerequires transcription of early genes via the virus-encoded RNApolymerases that are packaged in virions. However, this deficiency canbe overcome if virus DNA is transfected into cells previously infectedwith a helper poxvirus, providing the necessary factors needed totranscribe, replicate, and package the transfected genome in trans (SamC K, Dumbell K R. Expression of poxvirus DNA in coinfected cells andmarker rescue of thermosensitive mutants by subgenomic fragments of DNA.Ann Virol (Inst Past). 1981; 132:135-50). Although this produces mixedviral progeny, the problem can be overcome by performing thereactivation reaction in a cell line that supports the propagation ofboth viruses, and then eliminating the helper virus by plating themixture of viruses on cells that do not support the helper virus' growth(Scheiflinger F, Dorner F, Falkner F G. Construction of chimericvaccinia viruses by molecular cloning and packaging. Proceedings of theNational Academy of Sciences of the United States of America. 1992;89(21):9977-81).

Previously, a method where the high-frequency recombination reactionscatalyzed by a Leporipoxvirus, Shope fibroma virus (SFV), can be coupledwith an SFV-catalyzed reactivation reaction, to rapidly assemblerecombinant VACV strains using multiple overlapping fragments of viralDNA (Yao X D, Evans D H. High-frequency genetic recombination andreactivation of orthopoxviruses from DNA fragments transfected intoleporipoxvirus-infected cells. Journal of Virology. 2003;77(13):7281-90). For the first time, the reactivation andcharacterization of a functional poxvirus (synthetic chimeric horsepoxvirus [scHPXV]) using chemically synthesized, overlappingdouble-stranded DNA fragments is described. The principles can beanalogously applied and extrapolated to other poxviruses, including butnot limited to camelpox virus (CMLV), cowpox virus (CPXV), ectromeliavirus (ECTV, “mousepox agent”), horsepox (HPXV), monkeypox virus (MPXV),rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus, Taterapoxvirus, Uasin Gishu disease virus, vaccinia virus (VACV), and volepoxvirus (VPV).

It is further shown here that one embodiment of a synthetic chimericpoxvirus of the invention (e.g., a synthetic chimeric horsepox virus),can infect and immunize mice against a lethal VACV challenge and can doso without causing any disease during the initial immunization step.

Synthetic Chimeric Poxviruses of the Invention

The invention provides functional synthetic chimeric poxviruses (scPVs)that are initially replicated and assembled from chemically synthesizedDNA. The viruses that may be produced in accordance with the methods ofthe invention can be any poxvirus whose genome has been sequenced inlarge part or for which a natural isolate is available. An scPV of theinvention may be based on the genome sequences of naturally occurringstrains, variants or mutants, mutagenized viruses or geneticallyengineered viruses. The viral genome of an scPV of the inventioncomprises one or more modifications relative to the wild type genome orbase genome sequence of said virus. The modifications may include one ormore deletions, insertions, substitutions, or combinations thereof. Itis understood that the modifications may be introduced in any number ofways commonly known in the art. The modified portions of the genome maybe derived from chemically synthesized DNA, cDNA or genomic DNA.

Chemical genome synthesis is particularly useful when a natural templateis not available for genetic modification, amplification, or replicationby conventional molecular biology methods. For example, a naturalisolate of horsepox virus (HPXV) is not readily available to obtaintemplate DNA but the genome sequence for HPXV (strain MNR-76) has beendescribed. The HPXV genome sequence, however, is incomplete. Thesequence of the terminal hairpin loops was not determined. In asurprising result, a functional synthetic chimeric HPXV (scHPXV) wasgenerated by using terminal hairpin loops based on VACV telomeres inlieu of HPXV terminal hairpin loop sequences. In some embodiments, thepoxvirus belongs to the Chordopoxvirinae subfamily. In some embodiments,the poxvirus belongs to a genus of Chordopoxvirinae subfamily selectedfrom Avipoxvirus, Capripoxvirus, Cervidpoxvirus, Crocodylipoxvirus,Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Parapoxvirus,Suipoxvirus, or Yatapoxvirus. In some embodiments, the poxvirus is anOrthopoxvirus. In some embodiments, the Orthopoxvirus is selected fromcamelpox virus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV,“mousepox agent”), HPXV, monkeypox virus (MPXV), rabbitpox virus (RPXV),raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu diseasevirus, vaccinia virus (VACV), variola virus (VARV) and volepox virus(VPV). In a preferred embodiment, the poxvirus is an HPXV. In anotherpreferred embodiment, the poxvirus is a VACV. In some embodiments, thepoxvirus is a Parapoxvirus. In some embodiments, the Parapoxvirus isselected from orf virus (ORFV), pseudocowpox virus (PCPV), bovinepopular stomatitis virus (BPSV), squirrel parapoxvirus (SPPV), red deerparapoxvirus, Ausdyk virus, Chamois contagious ecythema virus, reindeerparapoxvirus, or sealpox virus. In some embodiments, the poxvirus is aMolluscipoxvirus. In some embodiments, the Molluscipoxvirus is molluscumcontagiousum virus (MCV). In some embodiments, the poxvirus is aYatapoxvirus. In some embodiments, the Yatapoxvirus is selected fromTanapox virus or Yaba monkey tumor virus (YMTV). In some embodiments,the poxvirus is a Capripoxvirus. In some embodiments, the Capripoxvirusis selected from sheepox, goatpox, or lumpy skin disease virus. In someembodiments, the poxvirus is a Suipoxvirus. In some embodiments, theSuipoxvirus is swinepox virus. In some embodiments, the poxvirus is aLeporipoxvirus. In some embodiments, the Leporipoxvirus is selected frommyxoma virus, Shope fibroma virus (SFV), squirrel fibroma virus, or harefibroma virus. New poxviruses (e.g., Orthopoxviruses) are still beingconstantly discovered. It is understood that an scPV of the inventionmay be based on such a newly discovered poxvirus.

Chemical viral genome synthesis also opens up the possibility ofintroducing a large number of useful modifications to the resultinggenome or to specific parts of it. The modifications may improve ease ofcloning to generate the virus, provide sites for introduction ofrecombinant gene products, improve ease of identifying reactivated viralclones and/or confer a plethora of other useful features (e.g.,introducing a desired antigen, producing an oncolytic virus, etc.). Insome embodiments, the modifications may include the attenuation ordeletion of one or more virulence factors. In some embodiments, themodifications may include the addition or insertion of one or morevirulence regulatory genes or gene-encoding regulatory factors.

Traditionally, the terminal hairpins of poxviruses have been difficultto clone and sequence, hence, it is not surprising that some of thepublished genome sequences (e.g., VACV, ACAM 2000 and HPXV MNR-76) areincomplete. The published sequence of the HPXV genome is likewiseincomplete, probably missing ˜60 bp from the terminal ends. Thus, theHPXV hairpins cannot be precisely replicated and prior to thisinvention, it was not known whether HPXV could be replicated andassembled from polynucleotides based on only the known portion of theHPXV genome. Nor was it known that hairpins from one virus would beoperable in another. In an exemplary embodiment, 129 nt ssDNA fragmentswere chemically synthesized using the published sequence of the VACVtelomeres as a guide and ligated onto dsDNA fragments comprising leftand right ends of the HPXV genome. In some embodiments, the terminalhairpins of an scPV of the invention are derived from VACV. In someembodiments, the terminal hairpins are derived from CMLV, CPXV, ECTV,HPXV, MPXV, RPXV, raccoonpox virus, skunkpox virus, Taterapox virus,Uasin Gishu disease virus or VPV. In some embodiments, the terminalhairpins are based on the terminal hairpins of any poxvirus whose genomehas been completely sequenced or a natural isolate of which is availablefor genome sequencing.

In some embodiments, the modifications may include the deletion of oneor more restriction sites. In some embodiments, the modifications mayinclude the introduction of one or more restriction sites. In someembodiments, the restriction sites to be deleted from the genome oradded to the genome may be selected from one or more of restrictionsites such as but not limited to AanI, AarI, AasI, AatI, AatII, AbaSI,AbsI, Acc65I, AccI, AccII, AccIII, Acil, AclI, AcuI, AfeI, AflII,AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI,AscI, AseI, AsiSI, AvaI, AvaII, AvrIl, BaeGI, BaeI, BamHI BanI, BanII,BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcoDI, BfaI, BfuAI,BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI,BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI,BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I,BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFαI, BsrGI,BsrI, BssHII, BssSαI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI,BstZ17I, Bsu36I, BtgI, BtgZI, BtsαI, BtsCI, BtsIMutI, Cac8I, ClaI,CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DrdI, EaeI,EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI,FauI, Fnu4HI, FokI, FseI, FspEI, FspI, HaeII, HaeIII, HgaI, HhaI,HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I,Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, I-Ceul, I-SceI,KasI, KpnI, LpnPI, MboI, MboII, MfeI, MluCI, MluI, MlyI, MmeI, MnlI,MscI, MseI, MseI, MslI, MspA1I, MspI MspJI, MwoI, NaeI, NarI, NciI,NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI,NspI, PacI, PaeR7I, PciI, PflFI, PflMI, PleI, PluTI, PmeI, PmlI, PpuMI,PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI,SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScrFI, SexAI, SfaNI, SfcI,SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SrfI, SspI, StuI,StyD4I, StyI, SwaI, TaqαI, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I,XbaI, XcmI, XhoI, XmaI, XmnI, or ZraI. It is understood that any desiredrestriction site(s) or combination of restriction sites may be insertedinto the genome or mutated and/or eliminated from the genome. In someembodiments, one or more AarI sites are deleted from the viral genome.In some embodiments, one or more BsaI sites are deleted from the viralgenome. In some embodiments, one or more restriction sites arecompletely eliminated from the genome (e.g., all the AarI sites in theviral genome may be eliminated). In some embodiments, one or more AvaIrestriction sites are introduced into the viral genome. In someembodiments, one or more StuI sites are introduced into the viralgenome. In some embodiments, the one or more modifications may includethe incorporation of recombineering targets including but not limited toloxP or FRT sites.

In some embodiments, the modifications may include the introduction offluorescence markers such as but not limited to green fluorescentprotein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyan/bluefluorescent protein (BFP), red fluorescent protein (RFP), or variantsthereof, etc.; selectable markers such as but not limited to drugresistance markers (e.g., E. coli xanthine-guanine phosphoribosyltransferase gene (gpt), Streptomyces alboniger puromycinacetyltransferase gene (pac), neomycin phosphotransferase I gene (nptI),neomycin phosphotransferase gene II (nptII), hygromycinphosphotransferase (hpt), sh ble gene, etc.; protein or peptide tagssuch as but not limited to MBP (maltose-binding protein), CBD(cellulose-binding domain), GST (glutathione-S-transferase), poly(His),FLAG, V5, c-Myc, HA (hemagglutinin), NE-tag, CAT (chloramphenicol acetyltransferase), DHFR (dihydrofolate reductase), HSV (Herpes simplexvirus), VSV-G (Vesicular stomatitis virus glycoprotein), luciferase,protein A, protein G, streptavidin, T7, thioredoxin, Yeast 2-hybrid tagssuch as B42, GAL4, LexA, or VP16; localization tags such as an NLS-tag,SNAP-tag, Myr-tag, etc. It is understood that other selectable markersand/or tags known in the art may be used. In some embodiments, themodifications include one or more selectable markers to aid in theselection of reactivated clones (e.g., a fluorescence marker such asYFP, a drug selection marker such as gpt, etc.) to aid in the selectionof reactivated viral clones. In some embodiments, the one or moreselectable markers are deleted from the reactivated clones after theselection step.

The scPVs of the invention can be used as vaccines to protect againstpathogenic poxviral infections (e.g., VARV, MPXV, MCV, ORFV, Ausdykvirus, BPSV, sealpox virus etc.), as therapeutic agents to treat orprevent pathogenic poxviral infections (e.g., VARV, MPXV, MCV, ORFV,Ausdyk virus, BPSV, sealpox virus etc.), as vehicles for heterologousgene expression, or as oncolytic agents. In some embodiments, the scPVsof the invention can be used as vaccines to protect against VARVinfection. In some embodiments, the scPVs of the invention can be usedto treat or prevent VARV infection.

Methods of Producing Synthetic Chimeric Poxviruses

The invention provides systems and methods for synthesizing,reactivating and isolating functional synthetic chimeric poxviruses(scPVs) from chemically synthesized overlapping double-stranded DNAfragments of the viral genome. Recombination of overlapping DNAfragments of the viral genome and reactivation of the functional scPVare carried out in cells previously infected with a helper virus.Briefly, overlapping DNA fragments that encompass all or substantiallyall of the viral genome of the scPV are chemically synthesized andtransfected into helper virus-infected cells. The transfected cells arecultured to produce mixed viral progeny comprising the helper virus andreactivated scPV. Next, the mixed viral progeny are plated on host cellsthat do not support the growth of the helper virus but allow thesynthetic chimeric poxvirus to grow, in order to eliminate the helpervirus and recover the synthetic chimeric poxvirus. In some embodiments,the helper virus does not infect the host cells. In some embodiments,the helper virus can infect the host cells but grows poorly in the hostcells. In some embodiments, the helper virus grows more slowly in thehost cells compared to the scPV.

In some embodiments, substantially all of the synthetic chimericpoxviral genome is derived from chemically synthesized DNA. In someembodiments, about 40%, about 50%, about 60%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, over 99%, or 100% of the synthetic chimeric poxviralgenome is derived from chemically synthesized DNA. In some embodiments,the poxviral genome is derived from a combination of chemicallysynthesized DNA and naturally occurring DNA.

The number of overlapping DNA fragments used in the methods of theinvention will depend on the size of the poxviral genome. Practicalconsiderations such as reduction in recombination efficiency as thenumber of fragments increases on the one hand, and difficulties insynthesizing very large DNA fragments as the number of fragmentsdecreases on the other hand, will also inform the number of overlappingfragments used in the methods of the invention. In some embodiments, thesynthetic chimeric poxviral genome may be synthesized as a singlefragment. In some embodiments, the synthetic chimeric poxviral genome isassembled from 2-14 overlapping DNA fragments. In some embodiments, thesynthetic chimeric poxviral genome is assembled from 4-12 overlappingDNA fragments. In some embodiments, the synthetic chimeric poxviralgenome is assembled from 6-10 overlapping DNA fragments. In someembodiments, the synthetic chimeric poxviral genome is assembled from8-12 overlapping DNA fragments. In some embodiments, the syntheticchimeric poxviral genome is assembled from 8-10, 10-12, or 10-14overlapping DNA fragments. In some embodiments, the synthetic chimericpoxviral genome is assembled from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 overlapping DNA fragments. In some embodiments, thesynthetic chimeric poxviral genome is assembled from 10 overlapping DNAfragments. In an exemplary embodiment of the disclosure, a syntheticchimeric horsepox virus (scHPXV) is reactivated from 10 chemicallysynthesized overlapping double-stranded DNA fragments. In someembodiments, terminal hairpin loops are synthesized separately andligated onto the fragments comprising the left and right ends of thepoxviral genome. In some embodiments, terminal hairpin loops may bederived from a naturally occurring template. In some embodiments, theterminal hairpins of an scPV of the invention are derived from VACV. Insome embodiments, the terminal hairpins are derived from CMLV, CPXV,ECTV, HPXV, MPXV, RPXV, raccoonpox virus, skunkpox virus, Taterapoxvirus, Uasin Gishu disease virus or VPV. In some embodiments, theterminal hairpins are based on the terminal hairpins of any poxviruswhose genome has been completely sequenced or a natural isolate of whichis available for genome sequencing. In some embodiments, all of thefragments encompassing the poxviral genome are chemically synthesized.In some embodiments, one or more of the fragments are chemicallysynthesized and one or more of the fragments are derived from naturallyoccurring DNA (e.g., by PCR amplification or by well-establishedrecombinant DNA techniques).

The size of the overlapping fragments used in the methods of theinvention will depend on the size of the poxviral genome. It isunderstood that there can be wide variations in fragment sizes andvarious practical considerations, such as the ability to chemicallysynthesize very large DNA fragments, will inform the choice of fragmentsizes. In some embodiments, the fragments range in size from about 2,000bp to about 50,000 bp. In some embodiments, the fragments range in sizefrom about 3,000 bp to about 45,000 bp. In some embodiments, thefragments range in size from about 4,000 bp to 40,000 bp. In someembodiments, the fragments range in size from about 5,000 bp to 35,000bp. In some embodiments, the largest fragments are about 20,000 bp,21,000 bp, 22,000 bp, 23,000 bp, 24,000 bp, 25,000 bp, 26,000 bp, 27,000bp, 28,000 bp, 29,000 bp, 30,000 bp, 31,000 bp, 32,000 bp, 33,000 bp,34,000 bp, 35,000 bp, 36,000 bp, 37,000 bp, 38,000 bp, 39,000 bp, 40,000bp, 41,000 bp, 42,000 bp, 43,000 bp, 44,000 bp, 45,000 bp, 46,000 bp,47,000 bp, 48,000 bp, 49,000 bp, or 50,000 bp. In an exemplaryembodiment of the disclosure, an scHPXV is reactivated from 10chemically synthesized overlapping double-stranded DNA fragments rangingin size from about 8,500 bp to about 32,000 bp (Table 1).

The helper virus may be any poxvirus that can provide the trans-actingenzymatic machinery needed to reactivate a poxvirus from transfectedDNA. The helper virus may have a different or narrower host cell rangethan an scPV to be produced (e.g., Shope fibroma virus (SFV) has a verynarrow host range compared to Orthopoxviruses such as vaccinia virus(VACV) or HPXV). The helper virus may have a different plaque phenotypecompared to the scPV to be produced. In some embodiments, the helpervirus is a Leporipoxvirus. In some embodiments, the Leporipoxvirus is anSFV, hare fibroma virus, rabbit fibroma virus, squirrel fibroma virus,or myxoma virus. In some embodiments, the helper virus is an SFV. Insome embodiments, the helper virus is an Orthopoxvirus. In someembodiments, the Orthopoxvirus is a camelpox virus (CMLV), cowpox virus(CPXV), ectromelia virus (ECTV, “mousepox agent”), HPXV, monkeypox virus(MPXV), rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus,Taterapox virus, Uasin Gishu disease virus, VACV and volepox virus(VPV). In some embodiments, the helper virus is an Avipoxvirus,Capripoxvirus, Cervidpoxvirus, Crocodylipoxvirus, Molluscipoxvirus,Parapoxvirus, Suipoxvirus, or Yatapoxvirus. In some embodiments, thehelper virus is a fowlpox virus. In some embodiments, the helper virusis an Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus.In some embodiments, the helper virus is a psoralen-inactivated helpervirus. In an exemplary embodiment of the disclosure, an scHPXV isreactivated from overlapping DNA fragments transfected into SFV-infectedBGMK cells. The SFV is then eliminated by plating the mixed viralprogeny on BSC-40 cells.

The skilled worker will understand that appropriate host cells to beused for the reactivation of the scPV and the selection and/or isolationof the scPV will depend on the particular combination of helper virusand chimeric poxvirus being produced by the methods of the invention.Any host cell that supports the growth of both the helper virus and thescPV may be used for the reactivation step and any host cell that doesnot support the growth of the helper virus may be used to eliminate thehelper virus and select and/or isolate the scPV. In some embodiments,the helper virus is a Leporipoxvirus and the host cells used for thereactivation step may be selected from rabbit kidney cells (e.g.,LLC-RK1, RK13, etc.), rabbit lung cells (e.g., R9ab), rabbit skin cells(e.g., SF1Ep, DRS, RAB-9), rabbit cornea cells (e.g., SIRC), rabbitcarcinoma cells (e.g., Oc4T/cc), rabbit skin/carcinoma cells (e.g.,CTPS), monkey cells (e.g., Vero, BGMK, etc.) or hamster cells (e.g.,BHK-21, etc.). In some embodiments, the helper virus is SFV.

The scPVs of the present invention can be propagated in any substratethat allows the virus to grow to titers that permit the uses of thescPVs described herein. In one embodiment, the substrate allows thescPVs to grow to titers comparable to those determined for thecorresponding wild-type viruses. The scPVs of the invention may be grownin cells (e.g., avian cells, bat cells, bovine cells, camel cells,canary cells, cat cells, deer cells, equine cells, fowl cells, gerbilcells, goat cells, human cells, monkey cells, pig cells, rabbit cells,raccoon cells, seal cells, sheep cells, skunk cells, vole cells, etc.)that are susceptible to infection by the poxviruses. Such methods arewell-known to those skilled in the art. Representative mammalian cellsinclude, but are not limited to BHK, BGMK, BRL3A, BSC-40, CEF, CEK, CHO,COS, CVI, HaCaT, HEL, HeLa cells, HEK293, human bone osteosarcoma cellline 143B, MDCK, NIH/3T3, Vero cells, etc.). For virus isolation, thescPV is removed from cell culture and separated from cellularcomponents, typically by well known clarification procedures, e.g., suchas gradient centrifugation and column chromatography, and may be furtherpurified as desired using procedures well known to those skilled in theart, e.g., plaque assays.

Polynucleotides of the Invention

The invention provides polynucleotides (e.g., double-stranded DNAfragments) for producing functional synthetic chimeric poxviruses(scPVs). The invention provides methods for producing functional scPVsfrom synthetic DNA (e.g., chemically synthesized DNA, PCR amplified DNA,engineered DNA, polynucleotides comprising nucleoside analogs, etc.). Insome embodiments, the invention provides methods for producingfunctional scPVs from chemically synthesized overlapping double-strandedDNA fragments of the viral genome. The polynucleotides of the inventionmay be designed based on publicly available genome sequences. Wherenatural isolates of a poxvirus are readily available, the viral genomemay be sequenced prior to selecting and designing the polynucleotides ofthe invention. Alternatively, where partial DNA sequences of a poxvirusare available, for example, from a clinical isolate, from a forensicsample or from PCR amplified DNA from material associated with aninfected person, the partial viral genome may be sequenced prior toselecting and designing the polynucleotides of the invention. An scPV ofthe invention, and thus, the polynucleotides of the invention, may bebased on the genome sequences of naturally occurring strains, variantsor mutants, mutagenized viruses or genetically engineered viruses.

The invention provides isolated polynucleotides including a nucleotidesequence that is at least 90% identical (e.g., at least 91%, 92%, 93%,or 94% identical), at least 95% identical (e.g., at least 96%, 97%, 98%,or 99% identical), or 100% identical to all or a portion of a referencepoxviral genome sequence or its complement. The isolated polynucleotidesof the invention may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000,30000, 35000, 40000, 45000 bp or more contiguous or non-contiguousnucleotides of a reference polynucleotide molecule (e.g., a referencepoxviral genome or a fragment thereof). One of ordinary skill in the artwill appreciate that nucleic acid sequences complementary to the nucleicacids, and variants of the nucleic acids are also within the scope ofthis invention. In further embodiments, the nucleic acid sequences ofthe invention can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In some aspects, the invention provides polynucleotides for producingscPVs wherein the poxvirus is selected from the genus Avipoxvirus,Capripoxvirus, Cervidpoxvirus, Crocodylipoxvirus, Leporipoxvirus,Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, orYatapoxvirus. In some embodiments, the poxvirus is an Orthopoxvirus. Insome embodiments, the Orthopoxvirus is selected from camelpox virus(CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, “mousepox agent”),HPXV, monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpox virus,skunkpox virus, Taterapox virus, Uasin Gishu disease virus, VACV,variola virus (VARV) and volepox virus (VPV). In a preferred embodiment,the poxvirus is an HPXV. In another preferred embodiment, the poxvirusis a VACV. In another preferred embodiment, the poxvirus is the ACAM2000clone of VACV. In another preferred embodiment, the poxvirus is the VACVstrain IOC (VACV-IOC) (Genbank Accession KT184690 and KT184691). Inanother preferred embodiment, the scVACV genome is based on ModifiedVaccinia virus Ankara (Genbank Acccession U94848; Genbank AccessionAY603355). In yet another preferred embodiment, the scVACV genome isbased on MVA-BN (Genbank Accession DQ983238). In some embodiments, thepoxvirus is a Parapoxvirus. In some embodiments, the Parapoxvirus isselected from orf virus (ORFV), pseudocowpox virus (PCPV), bovinepopular stomatitis virus (BPSV), squirrel parapoxvirus (SPPV), red deerparapoxvirus, Ausdyk virus, Chamois contagious ecythema virus, reindeerparapoxvirus, or sealpox virus. In some embodiments, the poxvirus is aMolluscipoxvirus. In some embodiments, the Molluscipoxvirus is molluscumcontagiousum virus (MCV). In some embodiments, the poxvirus is aYatapoxvirus. In some embodiments, the Yatapoxvirus is selected fromTanapox virus or Yaba monkey tumor virus (YMTV). In some embodiments,the poxvirus is a Capripoxvirus. In some embodiments, the Capripoxvirusis selected from sheepox, goatpox, or lumpy skin disease virus. In someembodiments, the poxvirus is a Suipoxvirus. In some embodiments, theSuipoxvirus is swinepox virus. In some embodiments, the poxvirus is aLeporipoxvirus. In some embodiments, the Leporipoxvirus is selected frommyxoma virus, Shope fibroma virus (SFV), squirrel fibroma virus, or harefibroma virus. New poxviruses (e.g., Orthopoxviruses) are still beingconstantly discovered. It is understood that an scPV of the inventionmay be based on such a newly discovered poxvirus.

In some aspects, the scPV is a CMLV whose genome is based on a publishedgenome sequence (e.g., strain CMS (Genbank Accession AY009089.1)). Insome aspects, the scPV is a CPXV whose genome is based on a publishedgenome sequence (e.g., strain Brighton Red (Genbank Accession AF482758),strain GRI-90 (Genbank Accession X94355)). In some aspects, the scPV isa ECTV whose genome is based on a published genome sequence (e.g.,strain Moscow (Genbank Accession NC_004105)). In some aspects, the scPVis a MPXV whose genome is based on a published genome sequence (e.g.,strain Zaire-96-1-16 (Genbank Accession AF380138)). In some aspects, thescPV is a RPXV whose genome is based on a published genome sequence(e.g. strain Utrecht (Genbank Accession AY484669)). In some aspects, thescPV is a Taterapox virus whose genome is based on a published genomesequence (e.g., strain Dahomey 1968 (Genbank Accession NC_008291)).

In one aspect, the invention provides polynucleotides for producing asynthetic chimeric horsepox virus (scHPXV). In a specific embodiment,the scHPXV genome may be based on the genome sequence described for HPXVstrain MNR-76 (SEQ ID NO: 49) (Tulman E R, Delhon G, Afonso C L, Lu Z,Zsak L, Sandybaev N T, et al. Genome of horsepox virus. Journal ofVirology. 2006; 80(18):9244-58). This genome sequence is incomplete andappears not to include the sequence of the terminal hairpin loops. It isshown here that terminal hairpin loops from vaccinia virus (VACV) can beligated onto the ends of the HPXV genome to produce functional scHPXVparticles using the methods of the invention. The HPXV genome may bedivided into 10 overlapping fragments as described in the workingexamples of the disclosure and shown in Table 1. In some embodiments,the genome may be divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 overlapping fragments. In some embodiments, the entire genomemay be provided as one fragment. The genomic locations of the exemplaryoverlapping fragments and fragment sizes are shown in Table 1. Table 2shows some of the modifications that may be made in these fragmentsrelative to the base sequence. The polynucleotides of the inventioncomprise nucleic acids sequences that are at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NOs: 1-10. In some embodiments, an isolated polynucleotide ofthe invention comprises a variant of these sequences, wherein suchvariants can include missense mutations, nonsense mutations,duplications, deletions, and/or additions. SEQ ID NO: 11 and SEQ ID NO:12 depict the nucleotide sequences of VACV (WR strain) terminal hairpinloops. In some embodiments, the terminal hairpin loops comprise nucleicacid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11 or SEQID NO: 12.

The invention provides isolated polynucleotides including a nucleotidesequence that is at least 90% identical (e.g., at least 91%, 92%, 93%,or 94% identical), at least 95% identical (e.g., at least 96%, 97%, 98%,or 99% identical), or 100% identical to all or a portion of a referenceHPXV genome sequence (e.g., SEQ ID NO: 49). In some embodiments, anisolated polynucleotide of the invention comprises a variant of thereference sequences, wherein such variants can include missensemutations, nonsense mutations, duplications, deletions, and/oradditions. The isolated polynucleotides of the invention may include atleast 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000 bp ormore contiguous or non-contiguous nucleotides of a referencepolynucleotide molecule (e.g., a reference HPXV genome including but notlimited to SEQ ID NO: 49, or a portion thereof).

In another aspect, the invention provides polynucleotides for producinga synthetic chimeric VACV (scVACV). In a specific embodiment, the scVACVgenome is based on a published VACV genome. In a specific embodiment,the scVACV genome is based on strain ACAM2000; Genbank AccessionAY313847). In a specific embodiment, the scVACV genome is based onVACV-IOC (Genbank Accession KT184690 and KT184691). In a specificembodiment, the scVACV genome is based on Modified Vaccinia virus Ankara(Genbank Acccession U94848; Genbank Accession AY603355). In a specificembodiment, the scVACV genome is based on MVA-BN (Genbank AccessionDQ983238). The VACV genome may be divided into 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 overlapping fragments. In some embodiments,the entire genome may be provided as one fragment. In a specificembodiment, the VACV genome is divided into the nine overlappingfragments as shown in Table 7. The polynucleotides of the inventioncomprise nucleic acids sequences that are at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NOs: 50-58. In some embodiments, an isolated polynucleotide ofthe invention comprises a variant of these sequences, wherein suchvariants can include missense mutations, nonsense mutations,duplications, deletions, and/or additions. In other embodiments, thescVACV genome is based on a VACV strain selected from Western Reserve(Genbank Accession NC 006998; Genbank Accession AY243312), CL3 (GenbankAccession AY313848), Tian Tian (Genbank Accession AF095689.1), Tian Tianclones TT9 (JX489136), TP3 (Genbank Accession KC207810) and TP5 (GenbankAccession KC207811), NYCBH, Wyeth, Copenhagen (Genbank AccessionM35027), Lister 107 (Genbank Accession DQ121394) Lister-LO (GenbankAccession AY678276), Modified Vaccinia virus Ankara (MVA) (GenbankAcccession U94848; Genbank Accession AY603355), MVA-BN (GenbankAccession DQ983238), Lederle, Tashkent clones TKT3 (Genbank AccessionKM044309) and TKT4 (KM044310), USSR, Evans, Praha, LIVP, Ikeda, IHD-W(Genbank Accession KJ125439), LC16m8 (AY678275), EM-63, IC, Malbran,Duke (Genbank Accession DQ439815), 3737 (Genbank Accession DQ377945),CV-1, Connaught Laboratories, CVA (Genbank Accession AM501482), Serro 2virus (Genbank Accession KF179385), Cantaglo virus isolate CM-01(Genbank Accession KT013210), Dryvax clones DPP15 (Genbank AccessionJN654981), DPP20 (Genbank Accession JN654985), DPP13 (Genbank AccessionJN654980), DPP17 (Genbank Accession JN654983), DPP21 (Genbank AccessionJN654986). The VACV genome may be divided into 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 overlapping fragments. In some embodiments,the entire genome may be provided as one fragment.

The invention provides in one embodiment isolated polynucleotidesincluding a nucleotide sequence that is at least 90% identical (e.g., atleast 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., atleast 96%, 97%, 98%, or 99% identical), or 100% identical to all or aportion of a reference genome sequence or its complement (e.g., VACV).In some embodiments, an isolated polynucleotide of the inventioncomprises a variant of the reference sequences, wherein such variantscan include missense mutations, nonsense mutations, duplications,deletions, and/or additions. The isolated polynucleotides of theinvention may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000,30000, 35000, 40000, 45000 bp or more contiguous or non-contiguousnucleotides of a reference genome or portion thereof.

Polynucleotides complementary to any of the polynucleotide sequencesdisclosed herein are also encompassed by the present invention.Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic or synthetic) or RNA molecules.RNA molecules include mRNA molecules. Additional coding or non-codingsequences may, but need not, be present within a polynucleotide of thepresent invention, and a polynucleotide may, but need not, be linked toother molecules and/or support materials.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Polynucleotides or variants may also, or alternatively, be substantiallyhomologous to a polynucleotide provided herein. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a polynucleotide of the invention (or its complement).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequences set forth in SEQ ID NOs: 1-10, or 50-58, orsequences complementary thereto. One of ordinary skill in the art willreadily understand that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6×SSCat room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ due to degeneracy in the geneticcode are also within the scope of the invention. For example, a numberof amino acids are designated by more than one triplet. Codons thatspecify the same amino acid, or synonyms (for example, CAU and CAC aresynonyms for histidine) may result in “silent” mutations which do notaffect the amino acid sequence of the protein. One skilled in the artwill appreciate that these variations in one or more nucleotides (up toabout 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among members of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

The present invention further provides recombinant cloning vectors andexpression vectors that are useful in cloning a polynucleotide of thepresent invention. The present invention further provides transformedhost cells comprising a polynucleotide molecule or recombinant vector ofthe invention, and novel strains or cell lines derived therefrom.

A host cell may be a bacterial cell, a yeast cell, a filamentous fungalcell, an algal cell, an insect cell, or a mammalian cell. In someembodiments, the host cell is E. coli. A variety of different vectorshave been developed for specific use in each of these host cells,including phage, high copy number plasmids, low copy number plasmids,and shuttle vectors, among others, and any of these can be used topractice the present invention.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pBAD18,pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18,mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectorssuch as pSA3 and pAT28. These and many other cloning vectors areavailable from commercial vendors such as BioRad, Strategene, andInvitrogen.

To aid in the selection of host cells transformed or transfected withcloning vectors of the present invention, the vector can be engineeredto further comprise a coding sequence for a reporter gene product orother selectable marker. Such a coding sequence is preferably inoperative association with the regulatory element coding sequences, asdescribed above. Reporter genes that are useful in the invention arewell-known in the art and include those encoding green fluorescentprotein, luciferase, xylE, and tyrosinase, among others. Nucleotidesequences encoding selectable markers are well known in the art, andinclude those that encode gene products conferring resistance toantibiotics or anti-metabolites, or that supply an auxotrophicrequirement. Examples of such sequences include those that encoderesistance to ampicillin, erythromycin, thiostrepton or kanamycin, amongmany others.

The vectors containing the polynucleotides of interest and/or thepolynucleotides themselves, can be introduced into the host cell by anyof a number of appropriate means, including electroporation,transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (e.g., where the vector is aninfectious agent such as vaccinia virus). The choice of introducingvectors or polynucleotides will often depend on features of the hostcell.

The present invention further provides transformed host cells comprisinga polynucleotide molecule or recombinant vector of the invention, andnovel strains or cell lines derived therefrom. In some embodiments, hostcells useful in the practice of the invention are E. coli cells. Astrain of E. coli can typically be used, such as e.g., E. coli TOP 10,or E. coli BL21 (DE3), DH5α, etc., available from the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110,USA and from commercial sources. In some embodiments, other prokaryoticcells or eukaryotic cells may be used. In some embodiments, the hostcell is a member of a genus selected from: Clostridium, Zymomonas,Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia,Candida, Pichia, or Saccharomyces. Such transformed host cells typicallyinclude but are not limited to microorganisms, such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA vectors, or yeast transformed with recombinant vectors, amongothers. Preferred eukaryotic host cells include yeast cells, althoughmammalian cells or insect cells can also be utilized effectively.Suitable host cells include prokaryotes (such as E. coli, B. subtillis,S. lividans, or C. glutamicum) and yeast (such as S. cerevisae, S.pombe, P. pastoris, or K. lactis).

In one aspect, the invention also includes the genome of the scPV, itsrecombinants, or functional parts thereof. A functional part of theviral genome may be a portion of the genome that encodes a protein orportion thereof (e.g., domain, epitope, etc.), a portion that comprisesregulatory elements or components of regulatory elements such as apromoter, enhancer, cis- or trans-acting elements, etc. Such viralsequences can be used to identify or isolate the virus or itsrecombinants, e.g., by using PCR, hybridization technologies, or byestablishing ELISA assays.

Exemplary Uses Prevention or Treatment of Pathogenic Poxviral Infections

The synthetic chimeric poxviruses (scPVs) of the invention can be usedin immunization of a subject against a pathogenic poxviral infection.The scPVs of the invention can be used to prevent, manage, or treat oneor more pathogenic poxviral infections in a subject. In someembodiments, the pathogenic poxvirus is an Orthopoxvirus (e.g., camelpoxvirus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, “mousepoxagent”), HPXV, monkeypox virus (MPXV), rabbitpox virus (RPXV),raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu diseasevirus, vaccinia virus (VACV), variola virus (VARV) and volepox virus(VPV)). In some embodiments, the pathogenic poxvirus is a Parapoxvirus(e.g., orf virus (ORFV), pseudocowpox virus (PCPV), bovine popularstomatitis virus (BPSV), squirrel parapoxvirus (SPPV), red deerparapoxvirus, Ausdyk virus, Chamois contagious ecythema virus, reindeerparapoxvirus, or sealpox virus). In some embodiments, the pathogenicpoxvirus is a Molluscipoxvirus (e.g., molluscum contagiousum virus(MCV)). In some embodiments, the pathogenic poxvirus is a Yatapoxvirus(e.g., Tanapox virus or Yaba monkey tumor virus (YMTV)). In someembodiments, the pathogenic poxvirus is a Capripoxvirus (e.g., sheepox,goatpox, or lumpy skin disease virus). In some embodiments, the poxvirusis a Suipoxvirus (e.g., swinepox virus). In some embodiments, thepathogenic poxvirus is a Leporipoxvirus (e.g., myxoma virus, Shopefibroma virus (SFV), squirrel fibroma virus, or hare fibroma virus). Insome embodiments, the pathogenic poxvirus is VARV. In some embodiments,the pathogenic poxvirus is MPXV. In some embodiments, the pathogenicpoxvirus is MCV. In some embodiments, the pathogenic poxvirus is ORFV.In some embodiments, the pathogenic poxvirus is CPXV. The pathogenicpoxvirus may be a poxvirus pseudotype or chimera. In some embodiments,the subject is a human subject. In some embodiments, the subject is ananimal subject. New poxviruses (e.g., Orthopoxviruses) are still beingconstantly discovered. It is understood that an scPV of the inventioncan be used in immunization of a subject against a newly discoveredpathogenic poxvirus or in the prevention, management, or treatment of aninfection by a newly discovered pathogenic poxvirus.

The scPVs of the invention can be used in immunogenic formulations,e.g., vaccine formulations. The formulations may be used to prevent,manage, neutralize, treat and/or ameliorate a pathogenic poxviralinfection. The immunogenic formulations may comprise either a live orinactivated scPV of the invention. The scPV can be inactivated bymethods well known to those of skill in the art. Common methods useformalin and heat for inactivation. In some embodiments, the immunogenicformulation comprises a live vaccine. Production of such liveimmunogenic formulations may be accomplished using conventional methodsinvolving propagation of the scPV in cell culture followed bypurification. For example, the scPV can be cultured in BHK, BGMK, BRL3A,BSC-40, CEF, CEK, CHO, COS, CVI, HaCaT, HEL, HeLa cells, HEK293, humanbone osteosarcoma cell line 143B, MDCK, NIH/3T3, Vero cells, etc., ascan be determined by the skilled worker.

In one aspect, the scPVs of the invention can be used to prevent,manage, or treat smallpox. The scPVs of the invention (e.g., a syntheticchimeric HPXV (scHPXV) or a synthetic chimeric VACV (scVACV)) can beused as a vaccine for the prevention of smallpox in individuals orpopulations that have been exposed, potentially exposed, or are at riskof exposure to smallpox. The scPVs of the invention can be used tocreate a new national stockpile of smallpox vaccine (e.g., an scHPXV orscVACV of the invention). In some embodiments, the scPVs of theinvention can be prophylactically administered to defense personnel,first responders, etc.

In one embodiment, a composition comprising a scHPXV of the invention isused as a smallpox vaccine. It is shown here that a scHPXV producedaccording to the methods of the invention has a small plaque phenotype.In general, a small plaque phenotype is considered to reflectattenuation. Accordingly, a scHPXV produced according to the methods ofthe invention provides a safe alternative to the existing smallpoxvaccines. In some embodiments, the vaccine may be safe foradministration to immunosuppressed subjects (e.g., HIV patients,patients undergoing chemotherapy, patients undergoing treatment forcancer, rheumatologic disorders, or autoimmune disorders, patients whoare undergoing or have received an organ or tissue transplant, patientswith immune deficiencies, children, pregnant women, patients with atopicdermatitis, eczema, psoriasis, heart conditions, and patients onimmunosuppressants etc.) who may suffer from severe complications froman existing smallpox vaccine and are thus contraindicated for anexisting smallpox vaccine. In some embodiments the vaccine may be usedin combination with one or more anti-viral treatments to suppress viralreplication. In some embodiments the vaccine may be used in combinationwith brincidofovir treatment to suppress viral replication. In someembodiments the vaccine may be used in combination withtecovirimat/SIGA-246 treatment to suppress viral replication. In someembodiments, the vaccine may be used in combination with acyclicnucleoside phosphonates (cidofovir), oral alkoxyalkyl prodrugs ofacyclic nucleoside or phosphonates (brincidofovir or CMX001). In someembodiments, the vaccine may be used in combination with Vaccinia ImmuneGlobulin (VIG). In some embodiments the vaccine may be used in subjectswho have been previously immunized with peptide or protein antigensderived from VACV, VARV or HPXV. In some embodiments the vaccine may beused in subjects who have been previously immunized with killed orinactivated VACV. In some embodiments the vaccine may be used insubjects who have been previously immunized with thereplication-deficient/defective VACV virus strain, MVA (modified virusAnkara). A vaccine formulation comprising a scHPXV of the invention maycomprise either a live or inactivated scHPXV.

In one embodiment, a composition comprising a scVACV of the invention isused as a smallpox vaccine. The scVACV may be based on a VACV strainselected from ACAM2000 (Genbank Accession AY313847), Western Reserve(Genbank Accession NC 006998; Genbank Accession AY243312), CL3 (GenbankAccession AY313848), Tian Tian (Genbank Accession AF095689.1), Tian Tianclones TT9 (JX489136), TP3 (Genbank Accession KC207810) and TP5 (GenbankAccession KC207811), NYCBH, Wyeth, Copenhagen (Genbank AccessionM35027), Lister 107 (Genbank Accession DQ121394) Lister-LO (GenbankAccession AY678276), Modified Vaccinia virus Ankara (MVA) (GenbankAcccession U94848; Genbank Accession AY603355), MVA-BN (GenbankAccession DQ983238), Lederle, Tashkent clones TKT3 (Genbank AccessionKM044309) and TKT4 (KM044310), USSR, Evans, Praha, LIVP, Ikeda, IHD-W(Genbank Accession KJ125439), LC16m8 (AY678275), EM-63, IC, Malbran,Duke (Genbank Accession DQ439815), 3737 (Genbank Accession DQ377945),CV-1, Connaught Laboratories, CVA (Genbank Accession AM501482), Serro 2virus (Genbank Accession KF179385), Cantaglo virus isolate CM-01(Genbank Accession KT013210), Dryvax clones DPP15 (Genbank AccessionJN654981), DPP20 (Genbank Accession JN654985), DPP13 (Genbank AccessionJN654980), DPP17 (Genbank Accession JN654983), DPP21 (Genbank AccessionJN654986) and IOC (Genbank Accession KT184690 and KT184691). In oneembodiment, the scVACV to be used as a smallpox vaccine is based onstrain ACAM2000 (Genbank Accession AY313847). In one embodiment, thescVACV to be used as a smallpox vaccine is based on strain VACV-IOC(Genbank Accession KT184690 and KT184691). In one embodiment, the scVAVCto be used as a smallpox vaccine is based on strain MVA (GenbankAcccession U94848; Genbank Accession AY603355). In one embodiment, thescVACV to be used as a smallpox vaccine is based on strain MVA-BN(Genbank Accession DQ983238). A vaccine formulation comprising a scPV ofthe invention may comprise either a live or inactivated scVACV.

In some embodiments, a composition comprising a scPV of the invention(e.g., a scHPXV or a scVACV) is used as a vaccine against a VACVinfection, a MPXV infection or a CPXV infection.

In some embodiments, a scPV of the invention may be designed to expressheterologous antigens or epitopes and can be used as vaccines againstthe source organisms of such antigens and/or epitopes.

The immunogenic formulations of the present invention (e.g., vaccines)comprise an effective amount of a scPV of the invention, and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeiae for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the pharmaceutical composition (e.g., immunogenic orvaccine formulation) is administered. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Examples of suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin. Theformulation should suit the mode of administration. The particularformulation may also depend on whether the scPV is live or inactivated.Purified scPVs of the invention may be lyophilized for later use or canbe immediately prepared in a pharmaceutical solution. The scPVs may alsobe diluted in a physiologically acceptable solution such as sterilesaline, with or without an adjuvant or carrier.

The immunogenic formulations (e.g., vaccines) of the invention may beadministered to patients by scarification. The vaccines may also beadministered by any other standard route of administration. Many methodsmay be used to introduce the immunogenic formulations (e.g., vaccines),these include but are not limited to intranasal, intratracheal, oral,intradermal, intramuscular, intraperitoneal, intravenous, conjunctivaland subcutaneous routes. In birds, the methods may further includechoanal inoculation. As an alternative to parenteral administration, theinvention also encompasses routes of mass administration foragricultural purposes such as via drinking water or in a spray.Alternatively, it may be preferable to introduce an scPV of theinvention via its natural route of infection. In some embodiments, theimmunogenic formulations of the invention are administered as aninjectable liquid, a consumable transgenic plant that expresses thevaccine, a sustained release gel or an implantable encapsulatedcomposition, a solid implant or a nucleic acid. The immunogenicformulation may also be administered in a cream, lotion, ointment, skinpatch, lozenge, or oral liquid such as a suspension, solution andemulsion (oil in water or water in oil).

In certain embodiments, an immunogenic formulation of the invention(e.g., vaccine) does not result in complete protection from aninfection, but results in a lower titer or reduced number of thepathogen (e.g., pathogenic poxvirus) compared to an untreated subject.In certain embodiments, administration of the immunogenic formulationsof the invention results in a 0.5 fold, 1 fold, 2 fold, 4 fold, 6 fold,8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold,125 fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold,750 fold, or 1,000 fold or greater reduction in titer of the pathogenrelative to an untreated subject. Benefits of a reduction in the titer,number or total burden of pathogen include, but are not limited to, lessseverity of symptoms of the infection and a reduction in the length ofthe disease or condition associated with the infection.

In certain embodiments, an immunogenic formulation of the invention(e.g., vaccine) does not result in complete protection from aninfection, but results in a lower number of symptoms or a decreasedintensity of symptoms, or a decreased morbidity or a decreased mortalitycompared to an untreated subject.

In various embodiments, the immunogenic formulations of the invention(e.g., vaccines) or antibodies generated by the scPVs of the inventionare administered to a subject in combination with one or more othertherapies (e.g., antiviral or immunomodulatory therapies) for theprevention of an infection (e.g., a pathogenic poxviral infection). Inother embodiments, the immunogenic formulations of the invention orantibodies generated by the scPVs of the invention are administered to asubject in combination with one or more other therapies (e.g., antiviralor immunomodulatory therapies) for the treatment of an infection (e.g.,a pathogenic poxviral infection). In yet other embodiments, theimmunogenic formulations of the invention or antibodies generated by thescPVs of the invention are administered to a subject in combination withone or more other therapies (e.g., antiviral or immunomodulatorytherapies) for the management and/or amelioration of an infection (e.g.,a pathogenic poxviral infection). In a specific embodiment, theimmunogenic formulations of the invention or antibodies generated by thescPVs of the invention are administered to a subject in combination withone or more other therapies (e.g., antiviral or immunomodulatorytherapies) for the prevention of smallpox. In another specificembodiment, the immunogenic formulations of the invention or antibodiesgenerated by the scPVs of the invention are administered to a subject incombination with one or more other therapies (e.g., antiviral orimmunomodulatory therapies) for the treatment of smallpox. In someembodiments the vaccine may be used in combination with one or moreanti-viral treatments to suppress viral replication. In some embodimentsthe vaccine may be used in combination with brincidofovir treatment tosuppress viral replication. In some embodiments the vaccine may be usedin combination with tecovirimat/SIGA-246 treatment to suppress viralreplication. In some embodiments, the vaccine may be used in combinationwith acyclic nucleoside phosphonates (cidofovir), oral alkoxyalkylprodrugs of acyclic nucleoside or phosphonates (brincidofovir orCMX001). In some embodiments, the vaccine may be used in combinationwith Vaccinia Immune Globulin (VIG). In some embodiments the vaccine maybe used in subjects who have been previously immunized with peptide orprotein antigens derived from VACV, VARV or HPXV. In some embodimentsthe vaccine may be used in subjects who have been previously immunizedwith killed or inactivated VACV. In some embodiments the vaccine may beused in subjects who have been previously immunized with thereplication-deficient/defective VACV virus strain, MVA (modified virusAnkara).

Any anti-viral agent well-known to one of skill in the art can be usedin the formulations (e.g., vaccine formulations) and the methods of theinvention. Non-limiting examples of anti-viral agents include proteins,polypeptides, peptides, fusion proteins antibodies, nucleic acidmolecules, organic molecules, inorganic molecules, and small moleculesthat inhibit and/or reduce the attachment of a virus to its receptor,the internalization of a virus into a cell, the replication of a virus,or release of virus from a cell. In particular, anti-viral agentsinclude but are not limited to antivirals that blocks extracellularvirus maturation (tecovirimat/SIGA-246), acyclic nucleoside phosphonates(cidofovir), oral alkoxyalkyl prodrugs of acyclic nucleosidephosphonates (brincidofovir or CMX001) or Vaccinia Immune Globulin(VIG). In some embodiments, anti-viral agents include, but are notlimited to, nucleoside analogs (e.g., zidovudine, acyclovir,gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin),foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, and AZT.

Doses and dosing regimens can be determined by one of skill in the artaccording to the needs of a subject to be treated. The skilled workermay take into consideration factors such as the age or weight of thesubject, the severity of the disease or condition being treated, and theresponse of the subject to treatment. A composition of the invention canbe administered, for example, as needed or on a daily basis. Dosing maytake place over varying time periods. For example, a dosing regimen maylast for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer. In someembodiments, a dosing regimen will last 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, or longer.

The scPVs of the invention can also be used to produce antibodies usefulfor passive immunotherapy, diagnostic or prognostic immunoassays, etc.Methods of producing antibodies are well-known in the art. Theantibodies may be further modified (e.g., chimerization, humanization,etc.) prior to use in immunotherapy.

Oncolytic Agents

The synthetic chimeric poxviruses (scPVs) of the invention can be usedas oncolytic agents that selectively replicate in and kill cancer cells.Cells that are dividing rapidly, such as cancer cells, are generallymore permissive for poxviral infection than non-dividing cells. Manyfeatures of poxviruses, such as safety in humans, ease of production ofhigh-titer stocks, stability of viral preparations, and capacity toinduce antitumor immunity following replication in tumor cells makepoxviruses desirable oncolytic agents. The scPVs produced according tothe methods of the invention may comprise one or modifications thatrender them suitable for the treatment of cancer. Accordingly, in oneaspect, the disclosure provides a method of inducing death in cancercells, the method comprising contacting the cells with an isolated scPVor pharmaceutical composition comprising an scPV of the invention. Inone aspect, the disclosure provides a method of treating cancer, themethod comprising administering to a patient in need thereof, atherapeutically effective amount of an scPV of the invention. Anotheraspect includes the use of an scPV or a composition described herein toinduce death in a neoplastic disorder cell such as a cancer cell or totreat a neoplastic disorder such as cancer. In some embodiments, thepoxvirus oncolytic therapy is administered in combination with one ormore conventional cancer therapies (e.g., surgery, chemotherapy,radiotherapy, thermotherapy, and biological/immunological therapy). Inspecific embodiments, the oncolytic virus is a synthetic chimeric VACV(scVACV) of this invention. In some embodiments, the oncotyic virus is asynthetic chimeric myxoma virus of this invention. In some embodiments,the oncolytic virus is a synthetic chimeric HPXV (scHPXV) of thisinvention. In some embodiments, the oncolytic virus is a syntheticchimeric raccoonpox virus of this invention. In some embodiments, theoncolytic virus is a synthetic chimeric yaba-like disease virus of thisinvention.

Using the methods of this invention, one or more desirable genes can beeasily introduced and one or more undesirable genes can be easilydeleted from the synthetic chimeric poxviral genome. In someembodiments, the scPVs of the invention for use as oncolytic agents aredesigned to express transgenes to enhance their immunoreactivity,antitumor targeting and/or potency, cell-to-cell spread and/or cancerspecificity. In some embodiments, an scPV of the invention is designedor engineered to express an immunomodulatory gene (e.g., GM-CSF, or aviral gene that blocks TNF function). In some embodiments, an scPV ofthe invention is designed to include a gene that expresses a factor thatattenuates virulence. In some embodiments, an scPV of the invention isdesigned or engineered to express a therapeutic agent (e.g., hEPO,BMP-4, antibodies to specific tumor antigens or portions thereof, etc.).In some embodiments, the scPVs of the invention have been modified forattenuation. In some embodiments, the scPV of the invention is designedor engineered to lack the viral TK gene. In some embodiments, an scVACVof the invention is designed or engineered to lack vaccinia growthfactor gene. In some embodiments, an scVACV of the invention is designedor engineered to lack the hemagglutinin gene.

The scPVs of the invention are useful for treating a variety ofneoplastic disorders and/or cancers. In some embodiments, the type ofcancer includes but is not limited to bone cancer, breast cancer,bladder cancer, cervical cancer, colorectal cancer, esophageal cancer,gliomas, gastric cancer, gastrointestinal cancer, head and neck cancer,hepatic cancer such as hepatocellular carcinoma, leukemia, lung cancer,lymphomas, ovarian cancer, pancreatic cancer, prostate cancer, renalcancer, skin cancer such as melanoma, testicular cancer, etc. or anyother tumors or pre-neoplastic lesions that may be treated.

In another embodiment, the method further comprises detecting thepresence of the administered scPV, in the neoplastic disorder or cancercell and/or in a sample from a subject administered an isolated orrecombinant virus or composition described herein. For example, thesubject can be tested prior to administration and/or followingadministration of the scPV or composition described herein to assess forexample the progression of the infection. In some embodiments, an scPVof the disclosure comprises a detection cassette and detecting thepresence of the administered chimeric poxvirus comprises detecting thedetection cassette encoded protein. For example, wherein the detectioncassette encodes a fluorescent protein, the subject or sample is imagedusing a method for visualizing fluorescence.

The oncolytic formulations of the present invention comprise aneffective amount of an scPV of the invention, and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeiae for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the pharmaceutical composition (e.g., oncolytic formulation) isadministered. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

Viral Vectors for Recombinant Gene Expression

The synthetic chimeric poxviruses (scPVs) of the invention may beengineered to carry heterologous sequences. The heterologous sequencesmay be from a different poxvirus species or from any non-poxviralsource. In one aspect, the heterologous sequences are antigenic epitopesthat are selected from any non-poxviral source. In some embodiments, therecombinant virus may express one or more antigenic epitopes from anon-poxviral source including but not limited to Plasmodium falciparum,mycobacteria, Bacillus anthracis, Vibrio cholerae, MRSA, rhabdovirus,influenza virus, viruses of the family of flaviviruses, paramyxoviruses,hepatitis viruses, human immunodeficiency viruses, or from virusescausing hemorrhagic fever, such as hantaviruses or filoviruses, i.e.,ebola or marburg virus. In another aspect, the heterologous sequencesare antigenic epitopes from a different poxvirus species. These viralsequences can be used to modify the host spectrum or the immunogenicityof the scPV.

In some embodiments, an scPV of the invention may code for aheterologous gene/nucleic acid expressing a therapeutic nucleic acid(e.g., antisense nucleic acid) or a therapeutic peptide (e.g., peptideor protein with a desired biological activity).

In some embodiments, the expression of a heterologous nucleic acidsequence is preferably, but not exclusively, under the transcriptionalcontrol of a poxvirus promoter. In some embodiments, the heterologousnucleic acid sequence is preferably inserted into a non-essential regionof the virus genome. Methods for inserting heterologous sequences intothe poxviral genome are known to a person skilled in the art. In someembodiments, the heterologous nucleic acid is introduced by chemicalsynthesis. In an exemplary embodiment, a heterologous nucleic acid maybe cloned into the HPXV095/J2R or HPXV044 locus of an scHPXV of theinvention.

An scPV of the present invention may be used for the introduction of aheterologous nucleic acid sequence into a target cell, the sequencebeing either homologous or heterologous to the target cell. Theintroduction of a heterologous nucleic acid sequence into a target cellmay be used to produce in vitro heterologous peptides or polypeptides,and/or complete viruses encoded by the sequence. This method comprisesthe infection of a host cell with the scPV; cultivation of the infectedhost cell under suitable conditions; and isolation and/or enrichment ofthe peptide, protein and/or virus produced by the host cell.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, andaccompanying embodiments.

EXAMPLES Example 1. Selection and Design of Overlapping Fragments of theViral Genome Materials and Methods

Synthetic Chimeric HPXV (scHPXV) Genome Design

Design of the scHPXV genome is based on the previously described genomesequence for HPXV (strain MNR-76; FIG. 1A) [GenBank accession DQ792504](Tulman E R, Delhon G, Afonso C L, Lu Z, Zsak L, Sandybaev N T, et al.Genome of horsepox virus. Journal of Virology. 2006; 80(18):9244-58).The 212,633 bp genome is divided into 10 overlapping fragments (FIG.1B). These fragments were designed so that they shared at least 1.0 kbpof overlapping sequence (i.e. homology) with each adjacent fragment, toprovide sites where homologous recombination will drive the assembly offull-length genomes (Table 1). These overlapping sequences will providesufficient homology to accurately carry out recombination between theco-transfected fragments (Yao X D, Evans D H. High-frequency geneticrecombination and reactivation of orthopoxviruses from DNA fragmentstransfected into leporipoxvirus-infected cells. Journal of Virology.2003; 77(13):7281-90). It is possible that shorter or longer overlapswill serve a similar purpose. The terminal 40 bp from the HPXV genomesequence (5′-TTTATTAAATTTTACTATTTATTTAGTGTCTAGAAAAAAA-3′) (SEQ ID NO:59) is not included in the synthesized inverted terminal repeat (ITR)fragments. Instead, a SapI restriction site is added at the 5′-terminus(GA_LITR) and 3′-terminus (GA_RITR) of the ITR fragments followed by aTGT sequence. These SapI restriction sites are used to ligate the VACVterminal hairpins onto the ITR fragments (described below).

Each fragment is chemically synthesized and subcloned into a plasmidusing terminal SfiI restriction sites on each fragment. To assist withsub-cloning these fragments, AarI and BsaI restriction sites aresilently mutated in all the fragments, except for the two ITR-encodingfragments (Table 2). The BsaI restriction sites in the two ITR-encodingfragments are not mutated, in case these regions contain nucleotidesequence-specific recognition sites that are important for efficient DNAreplication and concatamer resolution.

A yfp/gpt cassette under the control of a poxvirus early late promoteris introduced into the HPXV095/J2R locus within GA_Fragment_3) so thatreactivation of HPXV (scHPXV YFP-gpt::095) will be easy to visualizeunder a fluorescence microscope. The gpt locus also provides a potentialtool for selecting reactivated viruses using drug selection. HPXV095encodes the HPXV homolog of the non-essential VACV J2R gene and byco-transfecting Fragment_3 and other HPXV clones into SFV-infected BGMKcells, along with VACV DNA, a variety of hybrid viruses are recovered,validating the selection strategy (FIGS. 13A and 13B). Silent mutationsare also introduced into the HPXV044 (VACV^(WR)F4L) sequence(GA_Fragment_2) to create two unique restrictions sites withinGA_Fragment_2 (Table 3). In some embodiments, these unique restrictionsites may be used to rapidly introduce recombinant gene products (suchas but not limited to, selectable markers, fluorescent proteins,antigens, etc.) into GA_Fragment_2 prior to reactivation of HPXV.

TABLE 1 The HPXV genome fragments used in this study. The size of eachfragment and location within the HPXV genome are indicated. Locationwithin HPXV [DQ792504] Fragment Name Size (bp) (bp) GA_Left ITR (SEQ IDNO: 1) 10,095    41-10,135 GA_Fragment 1A (SEQ ID NO: 2) 16,257 8505-24,761 GA_Fragment 1B (SEQ ID NO: 3) 16,287  23764-40,050GA_Fragment 2 (SEQ ID NO: 4) 31,946 38,705-70,650 GA_Fragment 3 (SEQ IDNO: 5) 25,566 68,608-94,173 GA_Fragment 4 (SEQ ID NO: 6) 28,662 92,587-121,248 GA_Fragment 5 (SEQ ID NO: 7) 30,252 119,577-149,828GA_Fragment 6 (SEQ ID NO: 8) 30,000 147,651-177,650 GA_Fragment 7 (SEQID NO: 9) 28,754 176,412-205,165 GA_Right ITR (SEQ ID NO: 10) 8,484204,110-212,593

TABLE 2 Silent mutations created in scHPXV YFP-gpt::095 fragments toremove AarI and BsaI restriction sites from HPXV genome. Nucleotidechange in Mutation Restriction coding Location verified by endonucleasestrand of in HPXV whole GA_HPXV recognition HPXV HPXV genome genomeFragment site removed genome Gene [DQ792504] sequencing GA_Frag_1A BsaIA to G HPXV011a 11,228 ✓ GA_Frag_1B BsaI A to G HPXV025 27,845 ✓GA_Frag_2 BsaI A to G HPXV040 41,232 ✓ BsaI G to A HPXV059 56,775 ✓ BsaIG to A HPXV066 67,836 ✓ GA_Frag_3 BsaI G to A HPXV083 84,361 ✓ AarI T toC HPXV091 89,368 ✓ GA_Frag_4 BsaI T to C HPXV099 96,239 ✓ BsaI A to GHPXV099 96,437 ✓ BsaI A to G HPXV110 109,492 ✓ BsaI A to G HPXV111110,661 ✓ BsaI G to A HPXV111 110,840 ✓ GA_Frag_4 BsaI C to T HPXV119120,933 ✓ GA_Frag_5 GA_Frag_5 BsaI A to G HPXV123 123,035 ✓ BsaI T to CHPXV145 144,834 ✓ GA_Frag_5 BsaI T to C HPXV146d 149,727 ✓ GA_Frag_6GA_Frag_6 BsaI G to A HPXV178b 175,070 ✓ GA_Frag_7 BsaI G to A HPXV182180,573 ✓ BsaI A to G HPXV192 187,476 ✓ AarI G to A HPXV193 188,761 ✓BsaI C to T HPXV197 195,680 ✓ AarI T to C HPXV200 199,873 ✓

TABLE 3 Introduction of silent nucleotide mutations in the HPXV044 (VACVF4L) gene to create unique restriction endonuclease sites inGA_Fragment_2. Restriction Nucleotide change endonuclease in the HPXVLocation in HPXV gene site created coding strand HPXV genome HPXV044AvaI A to C 44,512 StuI A to C 45,061Synthesis of the Slow (S) and Fast (F) Forms of the Terminal HairpinLoops from VACV (Strain WR)

The Slow (S) and Fast (F) forms of the terminal hairpin loops from VACV(strain WR) are synthesized as 157nt ssDNA fragments (Integrated DNATechnologies; FIG. 2B). Through DNA synthesis, a 5′ overhang comprisedof three nucleotides is left at the end of each hairpin (5′-ACA; FIG.2C). The concatamer resolution site from the HPXV sequence [DQ792504] isalso synthesized in the terminal hairpin loops (FIG. 2B).

Digestion and Purification of scHPXV YFP-Gpt::095 Fragments

Synthetic HPXV fragments are digested with SfI overnight at 50° C. ThescHPXV ITR fragments are individually digested with SapI (ThermoFisherScientific) for 1 h, inactivated at 65° C. for 10 minutes, beforedigestion with SfiI overnight at 50° C. Approximately 1 U of FastAPalkaline phosphatase is added to the scHPXV YFP-gpt::095 ITR digestionsand incubated at 3TC for an additional 1 h. All scHPXV YFP-gpt::095fragments are subsequently purified using a QiaexII DNA cleanup kit(Qiagen). All scHPXV YFP-gpt::095 fragments are eluted from the QiaexIIsuspension in 10 mM Tris-HCl. DNA concentrations are estimated using aNanoDrop (ThermoFisher Scientific).

Results

Poxviruses catalyze very high-frequency homologous recombinationreactions that are inextricably linked to the process of virusreplication. Herein, it is demonstrated that large fragments ofchemically synthesized HPXV duplex DNA can be joined to form afunctional scHPXV genome using virus-catalyzed recombination andreplication reactions.

Using the published sequence of the HPXV genome (strain WNR-76), the212,633 bp genome is divided into 10-overlapping fragments (FIG. 2). Allof the BsaI and AarI sites in every fragment except the ITRs aremutated, in case sequence-specific sites within this region areunknowingly required for efficient genome replication and concatamerresolution. As described above, to facilitate the addition of theterminal hairpin loop structures from VACV onto the end of the ITRs, aSapI recognition site is included next to the left- and right-terminalend of both LITR and RITR fragments, respectively (FIG. 2A). These SapIsites are embedded within the flanking vector sequences, and the SapIenzyme cuts downstream of the site, outside of the recognition sequenceand in the HPXV DNA. Thus when DNA is cut with SapI, it leaves stickyends within the DNA copied from the HPXV sequence and thus permits theassembly of a precise sequence copy (through a subsequent ligation),containing no extraneous restriction sites. The other ends of the LITRand RITR fragments (the internal ends with respect to the genome map)are each bounded by SfiI recognition sites, as are both ends of theremaining HPXV fragments. All of these DNAs are supplied in a plasmidform for easy propagation. To prepare the internal fragments fortransfection into SFV-infected cells, these plasmids are digested withSfiI to release the plasmid from each scHPXV YFP-gpt::095 fragment (seebelow for how the LITR and RITR fragments are processed). Followingdigestion, each reaction is purified to remove any contaminating enzyme,but the plasmid is not removed from the digestion and is co-transfectedalongside each scHPXV YFP-gpt::095 fragment. This does not interferewith the reaction and is done to minimize the amount of DNA manipulationand possible fragmentation of these large DNA fragments.

While the reaction efficiency may be affected by the number oftransfected fragments, greater than or less than 10 overlappingfragments may be used in the methods of the invention. Without beingbound by theory, ˜15 fragments may represent a practical upper limitwithout further optimization of the reactivation reaction. The ideallower limit would be a single genome fragment, but in practice thetelomeres are most easily manipulated as more modest-sized fragments(e.g., ˜10 kb).

Example 2. Ligation of VACV F- and S-Terminal Hairpin Loops onto scHPXVYFP-Gpt::095 Left and Right ITR Fragments Materials and Methods

Ligation of the S- and F-Forms of the Terminal Hairpin Loops onto scHPXVYFP-Gpt::095 ITR Fragments

Approximately one microgram of each of the terminal VACV hairpin loopsis incubated at 95° C. for 5 minutes followed by a “snap” cool on ice toform the hairpin structure. The hairpin loops are subsequentlyphosphorylated at their 5′ end before ligation. Briefly, separate 20 μlreactions containing 1 μg of either VACV F-hairpin or VACV S-hairpin, 2μl of 10× T4 polynucleotide kinase buffer (ThermoFisher Scientific), 1mM ATP, and 10 units of T4 polynucleotide kinase (ThermoFisherScientific) are incubated at 37° C. for 1 h. The reaction is terminatedby heat inactivation at 75° C.

Approximately one microgram of either left ITR or right ITR is incubatedseparately with a 20-fold molar excess of each terminal hairpin in thepresence of 5% PEG-4000, and 5 units of T4 DNA ligase overnight at 16°C. Each ligation reaction is heat-inactivated at 65° C. for 10 minutesfollowed by incubation on ice until ready to transfect into cells.

Results

Orthopoxviruses encode linear dsDNA genomes bearing variable lengthinverted terminal repeats (ITR) at each end of the genome. The twostrands of the duplex genome are connected by hairpin loops to form acovalently continuous polynucleotide chain. The loops are A+T-rich,cannot form a completely base-paired structure, and exist in two formsthat are inverted and complementary in sequence (Baroudy B M, VenkatesanS, Moss B). Incompletely base-paired flip-flop terminal loops link thetwo DNA strands of the vaccinia virus genome into one uninterruptedpolynucleotide chain. Cell. 1982; 28(2):315-24) (FIG. 2B). They arecalled slow [S] and fast [F] forms based upon their electrophoreticproperties and probably fold into partially duplex hairpin structuresthat cap the ends of the linear dsDNA genome (FIG. 2C). The publishedsequence of the HPXV genome is incomplete, probably missing ˜60 bp fromthe terminal ends, making it impossible to precisely replicate the HPXVhairpins. Instead, 157 nt ssDNA fragments were chemically synthesizedusing the published sequence of the VACV telomeres as a guide andleaving a 5′ overhang comprised of three nucleotides at the end of eachhairpin (5′-ACA; FIG. 2C) (Baroudy B M, Venkatesan S, Moss B).Incompletely base-paired flip-flop terminal loops link the two DNAstrands of the vaccinia virus genome into one uninterruptedpolynucleotide chain. Cell. 1982; 28(2):315-24). This overhang iscomplementary to the ends generated by cutting cloned LITR and RITRfragments with SapI.

Sequences derived from VACV are used based upon data suggesting a closecommon ancestry between HPXV and VACV. It may be possible to use otherterminal hairpins from other poxviruses since there are sequencefeatures that are commonly conserved between the hairpin ends ofdifferent Chordopoxviruses. For example, the resolution sites in thehairpin ends are highly conserved in both sequence and functionality(they resemble late promoters).

These single-stranded oligonucleotides are heated to 95° C. and thenquickly chilled on ice to form the incompletely base-paired terminalhairpin (FIG. 2C). Next, each oligonucleotide is phosphorylated andligated separately at 20-fold molar excess with either the left or rightITR fragment previously digested with both SapI and SfiI. Digestion ofthe ITRs with these enzymes results in a 5′-TGT overhang at the 5′termini of each ITR, which was complementary to the 5′-ACA overhang inthe terminal hairpin loop structure. This produces a hairpin-terminatedcopy of each ITR.

To confirm that a hairpin-terminated structure is added to both ITRfragments, restriction digestion of the ITR fragments with PvuII isperformed. Since it is impossible to visualize the addition of a ˜70 bpterminal hairpin onto the terminus of a ˜10 kb ITR by gelelectrophoresis, a small amount of each ligation is digested with PvuII.If no terminal hairpin is ligated to the ITR, then digestion with PvuIIresults in a 1472 bp product (FIGS. 3A and 3B, lanes 2 and 5). If,however, the terminal hairpin loop is successfully added to the HPXVITRs, then an increase in the size of the ITR fragment is seen on anagarose gel (FIG. 3B, compare lane 2 with 3 and 4; compare lane 5 with 6and 7). These data suggest that under these conditions almost all of theHPXV ITRs contain terminal hairpin loops at one end of the fragment.

Example 3. Reactivation of scHPXV YFP-Gpt::095 from ChemicallySynthesized dsDNA Fragments Materials and Methods Viruses and CellCulture

SFV strain Kasza and BSC-40 were originally obtained from the AmericanType Culture Collection. Buffalo green monkey kidney (BGMK) cells wereobtained from G. McFadden (University of Florida). BSC-40 and BGMK cellsare propagated at 37° C. in 5% CO₂ in minimal essential medium (MEM)supplemented with L-glutamine, nonessential amino acids, sodiumpyruvate, antibiotics and antimycotics, and 5% fetal calf serum (FCS;ThermoFisher Scientific).

Reactivation of scHPXV YFP-Gpt::095 in Shope Fibroma Virus-InfectedCells

Buffalo green monkey kidney (BGMK) cells are grown in MEM containing 60mm tissue-culture dishes until they reached approximately 80%confluency. Cells are infected with Shope Fibroma Virus (SFV) inserum-free MEM at a MOI of 0.5 for 1 h at 37° C. The inoculum isreplaced with 3 ml of warmed MEM containing 5% FCS and returned to theincubator for an additional hour. Meanwhile, transfection reactions areset up as follows. Lipofectamine complexes are prepared by mixingapproximately 5 μg total synthetic HPXV DNA fragments in 1 ml Opti-MEMwith Lipofectamine2000 diluted in 1 ml Opti-MEM at a ratio of 3:1(Lipofectamine2000 to total DNA). A sample calculation to determine therelative amount of each HPXV fragment is shown in Table 4. The complexesare incubated at room temperature for 10 minutes and then added dropwiseto the BGMK cells previously infected with SFV. Approximately 16 h postinfection, the media is replaced with fresh MEM containing 5% FCS. Thecells are cultured for an additional 4 d (total of 5 d) at 37° C. Virusparticles were recovered by scraping the infected cells into the cellculture medium and performing three cycles of freezing and thawing. Thecrude extract is diluted 10⁻² in serum-free MEM and 4 ml of the inoculumis plated on 9-16 150 mm tissue culture plates of BSC-40 cells torecover reactivated scHPXV YFP-gpt::095. One hour post infection, theinoculum is replaced with MEM containing 5% FCS and 0.9% Noble Agar.Yellow fluorescent plaques are visualized under an inverted microscopeand individual plaques are picked for further analysis. ScHPXVYFP-gpt::095 plaques are plaque purified three times with yellowfluorescence selection.

TABLE 4 Sample calculation of the quantity of each GA_HPXV fragmenttransfected into SFV-infected BGMK cells. Ratio (frag. Amount of DNA tofrag. length:genome transfect (ng) fragment length length) ~1 μg ~3 μg~5 μg GA_LITR + F-hairpin 10,165 0.05 50 150 250 GA_LITR + S-hairpin10,165 0.05 50 150 250 GA_Frag_1A 16,257 0.08 80 240 400 GA_Frag_1B16,287 0.08 80 240 400 GA_Frag_2 31,946 0.15 150 450 750 GA_Frag_325,566 0.12 120 450 600 GA_Frag_4 28,662 0.13 130 390 650 GA_Frag_530,252 0.14 140 420 700 GA_Frag_6 30,000 0.14 140 420 700 GA_Frag_728,754 0.13 130 390 650 GA_RITR + F-hairpin 8,554 0.04 40 120 200GA_RITR + S-hairpin 8,554 0.04 40 120 200

Results

SFV-catalyzed recombination and reactivation of Orthopoxvirus DNA toassemble recombinant vaccinia viruses has previously been described (YaoX D, Evans D H. High-frequency genetic recombination and reactivation oforthopoxviruses from DNA fragments transfected intoleporipoxvirus-infected cells. Journal of Virology. 2003;77(13):7281-90; and Yao X D, Evans D H. Construction of recombinantvaccinia viruses using leporipoxvirus-catalyzed recombination andreactivation of orthopoxvirus DNA. Methods Mol Biol. 2004; 269:51-64).Several biological features make this an attractive model system. First,SFV has a narrow host range, productively infecting rabbit cells andcertain monkey cell lines, like BGMK. It can infect, but grows verypoorly on cells like BSC-40. Second, it grows more slowly compared toOrthopoxviruses, taking approximately 4-5 days to form transformed“foci” in monolayers of cells, a characteristic that is very differentfrom Orthopoxviruses, which produce plaques within 1-2 days in culture.This difference in growth between Leporipoxviruses and Orthopoxvirusesallows one to differentiate these viruses by performing the reactivationassays in BGMK cells and plating the progeny on BSC-40 cells. In someembodiments, other helper viruses (such as but not limited to fowlpoxvirus) may be used. In some embodiments, different cell combinations maybe used.

BGMK cells are infected with SFV at a MOI of 0.5 and then transfectedwith 5 μg of digested GA_HPXV fragments (Table 4) 2 h later. Five dayspost transfection all of the infectious particles are recovered by celllysis and re-plated on BSC-40 cells, which only efficiently supportgrowth of HPXV (or other Orthopoxviruses). The resulting reactivatedscHPXV YFP-gpt::095 plaques are visualized under a fluorescencemicroscope. The visualization is enabled by the yfp/gpt selectablemarker in the HPXV095/J2R locus within Frag_3 (FIG. 2A). Virus plaquesare detected in BSC-40 monolayers within 48 h of transfection. Theefficiency of recovering scHPXV YFP-gpt::095 is dependent on a number offactors, including DNA transfection efficiency, but ranges up to a fewPFU/μg of DNA transfected.

Example 4. Confirmation of scHPXV YFP-Gpt::095 Genome Sequence by PCRand Restriction Fragment Analysis Materials and Methods

PCR and Restriction Digestion Analysis of scHPXV

To rapidly confirm the presence of scHPXV YFP-gpt::095 in reactivatedplaque picks, PCR primers are designed to flank individual BsaI sitesthat were mutated in the scHPXV (Table 5). Genomic scHPXV YFP-gpt::095DNA is isolated from BSC-40 cells infected with scHPXV YFP-gpt::095 andused as a template. Genomic DNA from VACV-infected BSC-40 cells is usedas a control to confirm the presence of BsaI sites within each PCRproduct. Following PCR amplification, reactions are subsequentlydigested with BsaI for 1 h at 37° C. PCR reactions are separated on a 1%agarose gel containing SYBR® safe stain to visualize DNA bands.

Further analysis of scHPXV YFP-gpt::095 genomes by restriction digestionfollowed by pulse-field gel electrophoresis (PFGE) is carried out ongenomic DNA isolated using sucrose gradient purification (Yao X D, EvansD H. Construction of recombinant vaccinia viruses usingleporipoxvirus-catalyzed recombination and reactivation of orthopoxvirusDNA. Methods Mol Biol. 2004; 269:51-64). Briefly, 100 ng of purifiedviral genomic DNA is digested with 5 U of BsaI or HindIII for 2 h at 37°C. Digested DNA is run on a 1% Seakem Gold agarose gel cast and run in0.5× tris-borate-EDTA electrophoresis (TBE) buffer [110 mM tris; 90 mMborate; 2.5 mM EDTA]. The DNA is resolved on a CHEF DR-III apparatus(BioRad) at 5.7V/cm for 9.5 h at 14° C., using a switching time gradientof 1 to 10 s, a linear ramping factor, and a 120° angle. This programallows resolution of DNA species from 1 kbp to >200 kbp. To resolvefragments from 75 bp to 5 kbp, electrophoresis on 1.5% agarose gel castand run in 1.0×TBE at 115V for 2 h at room temperature is carried out.The DNA is visualized with SYBR® gold stain. The size of digested scHPXVYFP-gpt::095 DNA fragments is compared to control VACV genomic DNA.

TABLE 5Primers that are used in this study to amplify regions within VACV andHPXV surrounding the BsaI restriction sites found in GA_Fragment_1A,GA_Fragment_1B, GA_Fragment_2, GA_Fragment_3, GA_Fragment_4,GA_Fragment_5, GA_Fragment_6, and GA_Fragment_7. Position Positionof BsaI of BsaI site in site in VACV [NC_ HPXV Primer NamePrimer sequence (5 to 3′) 6998] [DQ792504] HPXV 1A-FWDCTGTATACCCATACTGAATTGATG  16,756  27,849 (SEQ ID NO: 13) AAC HPXV 1A-REVGAGTTAATATAGACGACTTTACT (SEQ ID NO: 14) AAAGTCATG HPXV 1B-FWDGGTTCTTTTTATTCTTTTAAACAG 23,076 N/A (SEQ ID NO: 15) ATCAATGG HPXV 1B-REVTTCTTATTAAGACATTGAGCCCAG (SEQ ID NO: 16) C HPXV 2A-FWDAGTCATCAATCATCATTTTTTCAC  30,073  41,225 (SEQ ID NO: 17) C HPXV 2A-REV(SEQ ID NO: 18) ATATAACGGACATTTCACCACC HPXV 2B-FWDGTAACATATACAACTTTTATTATG  45,485  56,778 (SEQ ID NO: 19) GCGTCHPXV 2B-REV CTAATCCACAAAAAATAGAATGT (SEQ ID NO: 20) TTAGTTATTTTGHPXV 2C-FWD AGTGACTGTATCCTCAAACATCC  56,576  67,839 (SEQ ID NO: 21)HPXV 2C-REV TTTATAAAGGGTTAACCTTTGTCA (SEQ ID NO: 22) CATC HPXV 3A-FWDTTGTGTAGCGCTTCTTTTTAGTC  60,981 N/A (SEQ ID NO: 23) HPXV 3A-REVAAACGGATCCATGGTAGAATATG (SEQ ID NO: 24) HPXV 3B-FWDTATTTGCATCTGCTGATAATCATC  84,916  84,353 (SEQ ID NO: 25) C HPXV 3B-REVCGATGGATTCAAATGACTTGTTA (SEQ ID NO: 26) ATG HPXV 4A-FWDATGCCTTTACAGTGGATAAAGTT  85,101  96,243 & (SEQ ID NO: 27) AAAC  96,428 HPXV 4A-REV CTGGATCCTTAGAGTCTGGAAG (SEQ ID NO: 28) HPXV 4B-FWDCGGAAAATGAAAAGGTACTAGAT  98,134 109,485 (SEQ ID NO: 29) ACG HPXV 4B-REVTGAATAGCCGTTAAATAATCTATT (SEQ ID NO: 30) TCGTC HPXV 4C-FWDTATGGATACATTGATAGCTATGA  99,302 & 110,653 & (SEQ ID NO: 31) AACG  99,481110,832 HPXV 4C-REV AATACATCTGTTAAAATTGTTTGA (SEQ ID NO: 32) CCCGHPXV 5A-FWD CATTTTATTTCTAGACGTTGCCAG 111,686 123,037 (SEQ ID NO: 33)HPXV 5A-REV CGATATGAAACTTCAGGCGG (SEQ ID NO: 34) HPXV 5B-FWDACAAAACGATTTAATTACAGAGT 122,484 N/A (SEQ ID NO: 35) TTTCAG HPXV 5B-REVGTCCGGTATGAGACGACAG (SEQ ID NO: 36) HPXV 5C-FWD TTAGGGATCACATGAATGAAATT133,505, 144,838 (SEQ ID NO: 37) CG HPXV 5C-REV TATGGAAGTTCCGTTTCATCCG(SEQ ID NO: 38) HPXV 5D-FWD GACTTGATAATCATATATTAAAC 138,306 149,718(SEQ ID NO: 39) ACATTGGATC HPXV 5D-REV AGATCTCCAGATTTCATAATATGA(SEQ ID NO: 40) TCAC HPXV 6A-FWD ATGATACGTACAATGATAATGAT 163,521 175,062(SEQ ID NO: 41) ACAGTAC HPXV 6A-REV TGATTTTTGCAATTGTCAGTTAAC(SEQ ID NO: 42) ACAAG HPXV 7A FWD TACTGTACCCACTATGAATAACG 169,035180,578 (SEQ ID NO: 43) C HPXV 7A-REV GATATCAACATCCACTGAAGAAG(SEQ ID NO: 44) AC HPXV 7B-FWD ATCTTACCATGTCCTCAAATAAAT 175,849 187,467(SEQ ID NO: 45) ACG HPXV 7B-REV ATAGCTCTAGGTATAGTCTGCAA (SEQ ID NO: 46)G HPXV 7C-FWD GCGAACTCCATTACACAAATATTT 181,952 195,683 (SEQ ID NO: 47) GHPXV 7D-REV GATGTTTCTAAATATAGGTTCCGT (SEQ ID NO: 48) AAGC

Results

The genome sequence of virus isolated from plaques grown from thereactivation assay is confirmed by PCR, restriction digestion, and wholegenome sequencing. The PCR analysis is based on the mutated BsaI siteswithin all but the ITR HPXV fragments. Primer sets are designed to flankeach BsaI site in scHPXV YFP-gpt::095 (Table 5). It is confirmed thatthese primer sets would also amplify a similar region within VACV WR.After PCR amplification of an approximate 1 kb region surrounding thesemutated BsaI sites within scHPXV YFP-gpt::095, each reaction is digestedwith BsaI and the resulting DNA fragments are analyzed by gelelectrophoresis. Since no BsaI sites are mutated in VACV (wt), enzymaticdigestion successfully digests each PCR product, resulting in a smallerDNA fragment (FIG. 4A, VACV). The PCR products generated from scHPXVYFP-gpt::095 genomic DNA are resistant to BsaI digestion, suggestingthat the BsaI recognition site is successfully mutated in these genomes(FIG. 4A, scHPXV YFP-gpt::095 (PP1) and scHPXV YFP-gpt::095 (PP3)). Theprimer products for primer set 7C did not result in any amplification ofDNA in the scHPXV YFP-gpt::095 PP1 and PP3 samples. To confirm whetherthis primer set was non-functional or if this area of Fragment 7 did notget assembled into the resulting scHPXV YFP-gpt::095 genome, PCR wasperformed on the original GA_Frag_7 plasmid DNA and this reaction wasalso unsuccessful in amplifying a product.

Genomic DNA is next isolated from sucrose-gradient purified scHPXVYFP-gpt::095 genomes, digested with BsaI or HindIII, and separated byagarose gel electrophoresis to confirm that the majority of the BsaIsites in scHPXV YFP-gpt::095 are successfully mutated. Interestingly,undigested genomic DNA from 3 different scHPXV YFP-gpt::095 clones runnoticeably slower on a gel compared to VACV, confirming that the genomeof scHPXV YFP-gpt::095 (213,305 bp) is larger than VACV-WR (194,711 bp)(FIG. 4B, compare lanes 2-4 with lane 5). The scHPXV YFP-gpt::095 clonesare resistant to BsaI digestion, resulting in one large DNA fragment(˜198000 bp) and a smaller DNA fragment at around 4000 bp afterseparation by PFGE (FIG. 4A, lane 7-9). This is in contrast to theVACV-WR genome, which when digested with BsaI, leads to a number of DNAfragments being separated on the gel (FIG. 4B, lane 10). Since theexpected DNA sizes following digestion of scHPXV YFP-gpt::095 genomewith BsaI are relatively small (Table 6), these digestion products areseparated by conventional agarose gel electrophoresis and it isconfirmed that the scHPXV YFP-gpt::095 generates the appropriate-sizedfragments (FIG. 4C, lanes 2-4). It is also confirmed that scHPXVYFP-gpt::095 produces the correct size of DNA fragments followingHindIII digestion, suggesting that these recognitions are maintainedduring synthesis of the large DNA fragments (Table 6; FIG. 4B, lanes12-14; FIG. 4C, lanes 6-8). Overall, in vitro analysis of the scHPXVYFP-gpt::095 genome suggests that reactivation of HPXV from chemicallysynthesized DNA fragments is successful.

TABLE 6 Expected sizes of scHPXV YFP-gpt::095 DNA fragments digestedwith either BsaI or HindIII. scHPXV YFP- scHPXV YFP- Fragment gpt::095digested gpt::095 digested # with BsaI (bp) with HindIII (bp) 1 198,83353,822 2 4046 24,848 3 4046 19,283 4 968 16,056 5 968 15,176 6 77813,836 7 778 13,558 8 767 12,679 9 767 8877 10 391 8637 11 391 6493 12138 5803 13 138 4631 14 64 4115 15 60 2216 16 54 1560 17 54 1442 18 32273 19 32

Since HPXV095 encodes the HPXV homolog of the non-essential VACV J2Rgene, by co-transfecting Fragment_3 and other HPXV clones intoSFV-infected BGMK cells, along with VACV DNA, a variety of hybridviruses are recovered, validating the selection strategy (FIGS. 13A and13B). The first hybrid virus (“VACV/HPXV+fragment 3”) is obtained byco-transfecting VACV DNA with HPXV Frag_3 (FIG. 1) into SFV-infectedcells. The green-tagged insertion encodes the YFP-gpt selection marker.Clones 1-3 are obtained by purifying the DNA from this first hybridgenome and transfecting it again, along with HPXV fragments 2, 4, 5, and7, into SFV-infected cells. PCR primers were designed to target bothHPXV and VACV (Table 5) are used to amplify DNA segments spanning theBsaI sites that are mutated in the scHPXV clones. Following PCRamplification, the products are digested with BsaI to differentiate VACVsequences (which cut) from HPXV (which do not cut). The VACV/HPXVhybrids exhibit a mix of BsaI sensitive and resistant sites whereas thereactivated scHPXV YFP-gpt::095 clone is fully BsaI resistant.

Example 5. Confirmation of scHPXV YFP-Gpt::095 Genome Sequence by WholeGenome Sequence Analysis Materials and Methods Virus DNA Isolation andSequencing

Stocks of HPXV YFP-gpt::095 clones (plaque pick [PP] 1.1, PP 2.1, and PP3.1]) are prepared and purified over sucrose gradients. Viral DNAs areextracted from each purified virus preparation using proteinase Kdigestion followed by phenol-chloroform extraction. The amount of dsDNAis determined using a Qubit dsDNA HS assay kit (ThermoFisherScientific). Each viral genome is sequenced at the Molecular BiologyFacility (MBSU) at the University of Alberta. Sequencing libraries aregenerated using the Nextera Tagmentation system (EpicentreBiotechnologies). Approximately 50 ng of each sample is sheared andlibrary prepped for paired end sequencing (2×300 bp) using an IlluminaMiSeq platform with an average read depth of 3,100 reads·nt⁻¹ across thegenome and ˜190 reads·nt⁻¹ in the F- and S-hairpins.

Sequence Assembly, Analysis, and Annotation

Raw sequencing reads are trimmed of low-quality sequence scores andinitially mapped to the HPXV reference sequence [GenBank AccessionDQ792504] using CLC Genomics Workbench 8.5 software. All nucleotideinsertions, deletions, and substitutions within the scHPXV YFP-gpt::095sequence are verified against the HPXV reference sequence. The GenomeAnnotation Transfer Utility (GATU) (Tcherepanov V, Ehlers A, Upton C.Genome Annotation Transfer Utility (GATU): rapid annotation of viralgenomes using a closely related reference genome. BMC Genomics. 2006;7:150. Epub 2006/06/15) is used to transfer the reference annotation tothe scHPXV genome sequences.

Results

Purified scHPXV YFP-gpt::095 genomes are sequenced using a multiplexapproach and an Illumina MiSeq sequencer. The sequence reads are mappedonto the wild-type HPXV (DQ792504) and scHPXV YFP-gpt::095 referencesequences to confirm the presence of specific modifications in thescHPXV YFP-gpt::095 genome. To confirm that the VACV terminal repeatsequences are correctly ligated onto the terminal end of the left ITR,sequencing reads in this area of the genome are analyzed. A string of Csis added to the beginning of the scHPXV YFP-gpt::095 genome referencesequence to capture all of the sequence reads that mapped in this region(FIGS. 5A and 5B). This is done because the program used to assemble thesequence reads will otherwise truncate the display of sequences at thepoint where the scHPXV YFP-gpt::095 genome reference sequence ends.

It is clear from the mapped reads that although the SapI recognitionsite is present in the scHPXV YFP-gpt::095 reference genome, all of thesequencing reads lack this sequence. This confirms that the approachdescribed herein produces an authentic HPXV sequence at the site wherethe synthetic hairpin was ligated to the ends of the ITRs. The completesequence of the VACV WR terminal hairpin loop is also successfullyobtained, which proves to be identical to the sequence of the syntheticssDNA that is ligated onto the TIR ends. Overall, these data suggestthat the VACV-WR terminal hairpin loops are successfully ligated ontothe HPXV ITR sequences and recovered in the infectious viruses.Moreover, the 1:1 distribution of F- and S-reads in each of five virusessuggested that both ends are required to produce a virus (FIG. 5B)

Next, it is verified that each nucleotide substitution to silentlymutate the BsaI sites has correctly been incorporated into the scHPXVYFP-gpt::095 genome (FIG. 6). Sequencing reads are mapped to the HPXV(DQ792504) reference sequence. The overall Illumina sequencing readcoverage in scHPXV YFP-gpt::095 from region 96,050 to 96,500 is shown inFIG. 6A. It is clear from here that there are two conflicts in thisregion that do not align correctly with reference HPXV (FIG. 6A, blueand yellow vertical lines). Upon magnification of these regions it isclear that at position 96,239 there is a T to C substitution (FIG. 6B)and at position 96,437 there is an A to G substitution (FIG. 6C) in thescHPXV YFP-gpt::095 genome. It was verified that all of the nucleotidesubstitutions that are introduced in order to mutate the selected BsaIand AarI recognition sites are created in the scHPXV YFP-gpt::095 genome(Table 2).

Finally, it is determined that the nucleotide substitutions in HPXV044,designed to create unique restriction sites in GA_Frag_2, are alsoincorporated into the scHPXV YFP-gpt::095 genome. The sequencing readsthat map to HPXV044 (region 44,400 to 45,100) are shown in FIG. 7A.Within this region there are two regions where the sequencing readsconflict with that of the sequence in the HPXV YFP-gpt::095 referencesequence (FIG. 7A, yellow vertical lines). Upon magnification of theseregions, it is clear that two T to G substitutions are introduced intothe non-coding strand of HPXV044 at positions 44,512 and 45,061, thuscreating AvaI and StuI restriction sites in Frag_2 (FIGS. 7B and 7C).Overall, the sequencing data corroborates the in vitro genomic analysisdata and confirms that scHPXV YFP-gpt::095 is successfully reactivatedin SFV-infected cells.

Example 6. ScHPXV YFP-Gpt::095 Replicates More Slowly in HeLa CellsCompared to Other Poxviruses Materials and Methods

BSC-40, HeLa, and HEL fibroblasts were originally obtained from theAmerican Type Culture Collection. BSC-40 cells are propagated at 37° C.in 5% CO₂ in minimal essential medium (MEM) supplemented withL-glutamine, nonessential amino acids, sodium pyruvate, antibiotics andantimycotics, and 5% fetal calf serum (FCS; ThermoFisher Scientific).HeLa and HEL cells are propagated at 37° C. in 5% CO₂ in Dulbecco'smodified Eagle's medium supplemented with L-glutamine, antibiotics andantimycotics, and 10% FCS.

Results

Multi-step growth curves and plaque size measurements are used toevaluate whether scHPXV YFP-gpt::095 replicated and spread in vitrosimilar to other Orthopoxviruses. Since a natural HPXV isolate isunavailable, the growth of scHPXV YFP-gpt::095 is compared to theprototypic poxvirus, VACV (strain WR), Cowpox virus (CPX), a poxvirusthat is closely related to HPXV and a clone of Dryvax virus, DPP15.Monkey kidney epithelial cells (BSC-40), Vero cells, a human carcinomacell line (HeLa), and primary human fibroblasts cells (HEL) are infectedwith VACV WR, CPX, DPP15, or scHPXV YFP-gpt::095 at a low MOI andinfected cells are harvested over a 72 h time course. In BSC-40 cells,the rate of virus replication and spread is comparable among all virusestested (FIG. 8A). Importantly, scHPXV YFP-gpt::095 replicates as well asany of the other poxviruses tested. The virus grew to somewhat lowertiters on HEL cells and Vero cells, and least well on HeLa cells. InHeLa cells, up to a 1.5-log decrease in virus production is seencompared to other Orthopoxviruses.

Next, the plaque size of scHPXV YFP-gpt::095 grown in BSC-40 cells ismeasured. A statistically significant decrease in plaque size of scHPXVYFP-gpt::095 compared to VACV WR and even cowpox virus (FIG. 8B) isobserved. Interestingly, in BSC-40 cells, scHPXV YFP-gpt::095 producesthe smallest plaques when compared to all other Orthopoxviruses tested(FIG. 8C). Also, while different VACV strains produce extracellularviruses that form smaller secondary plaques, these are not produced byscHPXV YFP-gpt::095 (FIG. 8C). Overall, these data suggest thatreactivation of scHPXV YFP-gpt::095 using the system described hereindoes not introduce any obvious defects in virus replication and spreadin vitro when compared to other Orthopoxviruses. Moreover, the plaquesize of scHPXV YFP-gpt::095 is similar to that of cowpox virus (CPXV),suggesting that synthetic virus reactivation does not have anydeleterious effects on the small plaque phenotype that has previouslybeen observed with other HPXV-like clones (Medaglia M L, Moussatche N,Nitsche A, Dabrowski P W, Li Y, Damon I K, et al. Genomic Analysis,Phenotype, and Virulence of the Historical Brazilian Smallpox VaccineStrain IOC: Implications for the Origins and Evolutionary Relationshipsof Vaccinia Virus. Journal of Virology. 2015; 89(23):11909-25).

Example 7. Removal of Yfp/Gpt Selection Marker

Following reactivation of the scHPXV YFP-gpt::095, the yfp/gpt selectionmarker in the HPXV095 locus is removed. To do this, a 1349 bp region ofsequence corresponding to nucleotide positions 91573 to 92921 in HPXV(DQ792504) is synthesized (ThermoFisher Scientific) (SEQ ID NO: 60).This fragment included approximately 400 bp of homology flanking eitherside of the wt HPXV095/J2R gene. This sequence of DNA is cloned into acommercial vector provided by GeneArt. To replace the yfp/gpt cassettewith the HPXV095 gene sequence, BSC-40 cells are infected with scHPXVYFP-gpt::095 at a MOI of 0.5 and then transfected, 2 h later, with 2 μgof linearized plasmid containing the wtHPXV095 sequence usingLipofectamine 2000 (ThermoFisher Scientific). The virus recombinants areharvested 48 h post infection and recombinant viruses (scHPXV (wt)) areisolated using three rounds of non-fluorescent plaque purification underagar. PCR is used to confirm the identity of the scHPXV (wt) usingprimers that flank the HPXV095 gene locus. The primers used to confirmthe correct replacement of the HPXV095 gene are HPXV095_check-FWD5′-CCTATTAGATACATAGATCCTCGTCG-3′ (SEQ ID NO: 61) and HPXV095_check-REV5′-CGGTTTATCTAACGACACAACATC-3′ (SEQ ID NO: 62).

Example 8. Growth Properties of scHPXV (Wt) Versus scHPXV YFP-Gpt::095

In experiments performed as described above in Example 6, scHPXV (wt)shows growth properties not significantly different from scHPXVYFP-gpt::095 in vitro (FIG. 14A-C). A statistically significant decreasein plaque size of scHPXV (wt) compared to VACV WR is observed (FIG.14A). scHPXV (wt), like scHPXV YFP-gpt::095, does not produceextracellular viruses (FIG. 14B) and there are no significantdifferences in the growth of scHPXV (wt) and scHPXV YFP-gpt::095 onBSC-40 cells, HEL cells, HeLA cells, and Vero cells (FIG. 14C). Thefinding that scHPXV (wt) does not produce extracellular viruses is ofrelevance given that this property affects virulence.

Example 9. Determination of the Virulence of scHPXV (Wt) in a MurineIntranasal Model

The toxicity effects of scHPXV (wt) are determined in this study. Forthis experiment, 6 groups of Balb/c mice are administered 3 differentdoses of scHPXV (ΔHPXV_095/J2R) or scHPXV (wt) described in Examples 1-7and compared to a PBS control group as well as a VACV (WR) control groupand a VACV (Dryvax strain DPP15) control group (9 treatment groups intotal). There are 3 additional mice included in this experiment that donot receive any treatment for the duration of the study. All mice aresampled for blood at predetermined points throughout the experiment andthe additional mice serve as a baseline for serum analysis.

Prior to inoculation of Balb/c mice, all virus strains are grown inBSC-40 cells (African green monkey kidney), harvested by trypsinization,washed in PBS, extracted from cells by dounce homogenization, purifiedthrough a 36% sucrose cushion by ultracentrifugation, resuspended inPBS, and titered such that the final concentrations are: 1) VACV(WR)—5×10⁵ PFU/ml; 2) VACV (DPP15)—10⁹ PFU/ml; 3) scHPXV(ΔHPXV_095/J2R)—10⁷ PFU/ml, 10⁸ PFU/ml, and 10⁹ PFU/ml and 4) scHPXV(wt)—10⁷ PFU/ml, 10⁸ PFU/ml, and 10⁹ PFU/ml.

The scHPXV doses chosen for this study (10⁵ PFU/dose, 10⁶ PFU/dose, and10⁷ PFU/dose) are based on previous studies using known vaccine strainsof VACV, including Dryvax and IOC (Medaglia M L, Moussatche N, NitscheA, Dabrowski P W, Li Y, Damon I K, et al. Genomic Analysis, Phenotype,and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC:Implications for the Origins and Evolutionary Relationships of VacciniaVirus. Journal of Virology. 2015; 89(23):11909-25; Qin L, Favis N,Famulski J, Evans D H. Evolution of and evolutionary relationshipsbetween extant vaccinia virus strains. Journal of Virology. 2015;89(3):1809-24).

Since weight loss is used as a measurement of virulence in mice, VACV(strain WR) is administered intranasally at a dose of 5×10³ PFU, whichleads to approximately 20-30% weight loss. The VACV Dryvax clone, DPP15,is also administered intranasally at 10⁷ PFU/dose, so that the virulenceof this well-known Smallpox vaccine can be directly compared to scHPXV(wt). Mice are purchased from Charles River Laboratories and oncereceived, are acclimatized to their environment for at least one weekprior to virus administration.

Each mouse receives a single dose of virus (˜10 ul) administered via theintranasal injection while under anesthesia. Mice are monitored forsigns of infection, such as swelling, discharge, or other abnormalitiesevery day for a period of 30 days. Each mouse is specifically monitoredfor weight loss every day after virus administration. Mice that losemore than 25% of their body weight in addition to other morbidityfactors are subjected to euthanasia in accordance with our animal healthcare facility protocols at the University of Alberta.

Even at the highest doses of scHPXV tested, there may be no overt signsof illness in Balb/c mice. The VACV strains most closely related toscHPXV, old South American viruses, in some cases produced no disease at10⁷ PFU (Medaglia ML, Moussatche N, Nitsche A, Dabrowski P W, Li Y,Damon I K, et al. Genomic Analysis, Phenotype, and Virulence of theHistorical Brazilian Smallpox Vaccine Strain IOC: Implications for theOrigins and Evolutionary Relationships of Vaccinia Virus. Journal ofVirology. 2015; 89(23):11909-25). It is impractical to test much higherdoses than this due to the difficulty of making purified stocks withtiters in excess of 10⁹ PFU/mL.

Example 10. Determination of Whether scHPXV Confers Immune ProtectionAgainst a Lethal VACV-WR Challenge

Mice that appear to have been unaffected by the initial virusadministration described in Example 9 continue to gain weight normallythroughout the experiment. Thirty days post virus inoculation, mice aresubsequently challenged with a lethal dose of VACV-WR (10⁶ PFU/dose) viaintranasal inoculation. Mice are closely monitored for signs ofinfection as described above. Mice are weighed daily and mice that losegreater than 25% of their body weight in addition to other morbidityfactors are subjected to euthanasia. We expect that mice inoculated withPBS prior to administration of a lethal dose of VACV-WR show signs ofsignificant weight loss and other morbidity factors within 7-10 dayspost inoculation. Approximately 14 days post lethal challenge withVACV-WR all mice are euthanized and blood is collected to confirm thepresence of VACV-specific neutralizing antibodies in the serum bystandard plaque reduction assays.

Example 11. Construction of a Synthetic Chimeric VACV (Strain ACAM2000)(scACAM2000) Using SFV-Catalyzed Recombination and ReactivationReactions Design of Overlapping Fragments of the VACV (ACAM2000) Genome

Using the published sequence of the VACV genome (strain ACAM2000;Genbank Accession AY313847), the genome is divided into 9 overlappingfragments (FIG. 9) that range in size from 15,979 bp to 28,795 bp inlength (Table 7). These fragments are designed so that they share atleast 1.0 kbp of overlapping sequence homology with each adjacentfragment to provide sites where homologous recombination can drive theassembly of full-length genomes (Table 7). These overlaps should besufficient to support accurate and efficient recombination between theco-transfected fragments.

In order to successfully synthesize and subclone these large fragments,each BsaI and AarI site in VACV_ACAM2000 fragments 1 to 7 are silentlymutated. As with the creation of scHPXV, the BsaI restriction sites inthe two ITR-encoding fragments are not mutated, in case there are DNAsequence features that were important for efficient DNA replication andconcatamer resolution.

For the initial reactivation, the thymidine kinase in VACV (ACAM2000) isreplaced with the yfp/gpt cassette to help recover newly reactivatedVACV particles.

TABLE 7 The VACV ACAM2000 genome fragments used in this study. The size,location within the VACV ACAM2000 genome [GenBank Accession AY313847],and overlap with adjacent fragments are described. Overlap with adjacentFragment Name Size (bp) Location (bp) fragment (bp) GA_LITR 18,073   1-18,073 — (A2000) Frag_1 (2287) (SEQ ID NO: 50) GA_Frag_1 24,89515,787-40,681 LITR (2287) (A2000) Frag_2 (1342) (SEQ ID NO: 51)GA_Frag_2 23,297 39,340-62,636 Frag_1 (1342) (A2000) Frag_3 (2453) (SEQID NO: 52) GA_Frag_3 24,971 60,184-85,154 Frag_2 (2453) (A2000) Frag_4(1604) (SEQ ID NO: 53) GA_Frag_4 26,575  83,551-110,125 Frag_3 (1604)(A2000) Frag_5 (1896) (SEQ ID NO: 54) GA_Frag_5 24,635 108,230-132,864Frag_4 (1896) (A2000) Frag_6 (2129) (SEQ ID NO: 55) GA_Frag_6 25,934130,736-156,669 Frag_5 (2129) (A2000) Frag_7 (2183) (SEQ ID NO: 56)GA_Frag_7 28,801 154,487-183,287 Frag_6 (2183) (A2000) RITR (1403) (SEQID NO: 57) GA_RITR 17,350 181,885-199,234 Frag_7 (1403) (A2000) — (SEQID NO: 58)Ligation of the S and F Forms of the Terminal Loops (from VACV StrainACAM2000) onto the Left and Right Ends of the VACV ITRs

To prepare the VACV ITR fragments to be transfected into SFV-infectedcells, the terminal hairpin loops are ligated to the left and right ITRfragments using the same methods used to attach VACV hairpins to theHPXV telomeres described. Briefly, through DNA synthesis a 5′ overhangcomprised of three nucleotides is left at the end of each hairpin(5′-ACA; as described in Examples 1 and 2). Meanwhile, the plasmidclones encoding the left and right VACV ITR fragments are designed toencode a SapI recognition site located immediately adjacent to the firstnucleotide encoding the start of the VACV genome. Digesting the ITRclone with SapI creates a three base overhang (5′-TGT), complementary tothe 5′-ACA overhang in the terminal hairpin loop structure. The left orright ITR fragments is mixed with a ˜20-fold molar excess of theterminal loops and ligated. This produces a hairpin-terminated copy ofeach ITR.

Leporipoxvirus-Catalyzed Recombination and Reactivation of scACAM2000

Following digestion and DNA clean up of the VACV genomic DNA fragments,they are transfected into SFV-infected BGMK cells. As describedpreviously, the SFV helper virus will catalyze the recombination betweenfragments sharing flanking homologous sequences, resulting in thecreation of full-length VACV genomes that can be packaged and releasedfrom the cell. It is unlikely that hybrid viruses will be produced inthis assay. After 4 days, the BGMK cells are harvested and reactivatedVACV virus particles are released by freeze-thaw, followed by plating onthe susceptible BSC-40 cells. The reactivated VACV (ACAM2000) plaquesare the only viruses to form plaques on BSC-40 cells. Plaques are pickedto produce clonal virus stocks followed by isolation of genomic DNA tobe sequenced by nextGen Illumina sequencing to confirm the integrity ofthe recovered viruses and identify whether scACAM2000 is successfullyreactivated.

Example 12. Safety and Immunogenicity of scHPXV in a Small Animal ModelMaterials and Methods

Murine Intranasal Model of scHPXV Infection.

Six-to-eight week old BALB/c mice are purchased from Charles RiverLaboratories. Groups of five mice are intranasally infected (or mockinfected) with 5×10³ PFU VACV (strain WR), 1×10⁷ VACV (Dryvax DPP15), or1×10⁵ PFU, 1×10⁶ PFU, or 1×10⁷ PFU of scHPXV YFP-gpt::095 (described inExamples 1-6) or scHPXV (wt) (described in Examples 7 and 8) in 10 μl ofPBS. The mice are weighed daily for 28 days, and the following clinicalsigns are scored: ruffled fur, difficulty breathing, reduced mobility,and pox lesions. Mice that lose 25% of the initial weight areeuthanized. These vaccinated mice are subsequently challengedintranasally with 1×10⁶ PFU VACV (strain WR), weighed and monitoreddaily for clinical signs of disease as described above for 13 days. Micethat lose 25% of the initial weight are euthanized as per protocolsapproved by the local animal care and use committee.

Results

scHPXV Strains do not Cause Weight Loss in an Intranasal Murine Model ofPoxvirus Infection

The toxicity effects of scHPXV YFP-gpt::095 or scHPXV (wt) are examinedin an intranasal murine model of poxvirus infection. Mice are inoculatedintranasally with the indicated dose(s) of scHPXV (wt) and their weightsare monitored over a 28-day period. No weight loss is apparent in miceinoculated with any of the doses of scHPXV YFP-gpt::095 or scHPXV (wt)over the 28-day period (FIG. 10). This is in contrast to animalsinoculated with either Dryvax DPP15 (10⁷ PFU) or VACV WR (5×10³ PFU),who lose an average of 15% and 10% of their initial weight,respectively. These data suggest that even at the highest dose of scHPXV(wt) and scHPXV YFP-gpt::095 tested (10⁷ PFU), no adverse effects areobserved. With a known smallpox vaccine strain, DPP15, however,transient weight loss is detected in mice by ˜7 days post inoculation,although these mice return to their initial weight by ˜10 days postinoculation.

scHPXV YFP-Gpt::095 (10⁶ & 10⁷ PFU) and scHPXV (Wt) (10⁵, 10⁶, 10⁷ PFU)Confer Immune Protection Against a Lethal VACV WR Challenge in BALB/cMice

Following the 28-day immunization of BALB/c mice with the indicatedstrains of scHPXV YFP-gpt::095, scHPXV (wt), DPP15, and VACV WR (FIG.10), mice are subsequently challenged with a lethal intranasal dose ofVACV WR (10⁶ PFU) to assess the protective efficacy of scHPXV (wt) orscHPXV YFP-gpt::095 in mice. All of the mice initially treated with PBSsuccumb to infection whereas the animals initially exposed to 10⁷ PFU ofDryvax (DPP15) or 5×10³ PFU VACV WR show no weight loss (FIG. 11A) andno signs of illness (FIG. 11B). Animals vaccinated with the two lowerdoses of the scHPXV YFP-gpt::095 show weight loss (FIG. 11A) and signsof severe illness based on clinical scores (FIG. 11B) and two animals inthe lowest scHPXV YFP-gpt::095 doses also are observed to succumb toinfection (FIG. 12). The remaining animals in these low-dose groups ofscHPXV YFP-gpt::095 are observed ultimately to recover from theinfection, but their weights remain lower than the average weights inthe rest of the groups. Animals previously exposed to 10⁵ to 10⁷ PFU ofscHPXV (wt) show only minor transient weight loss in the first few daysfollowing poxvirus challenge (FIG. 11A), and no clinical signs ofillness (FIG. 11B). These data show that scHPXV can infect and immunizemice against a lethal VACV challenge and can do so without causingdisease during the initial immunization step.

1. A synthetic chimeric poxvirus (scPV) that is replicated andreactivated from DNA derived from synthetic DNA, the viral genome ofsaid virus differing from a wild type genome of said virus in that it ischaracterized by one or more modifications, the modifications beingderived from a group comprising chemically synthesized DNA, cDNA orgenomic DNA.
 2. The scPV of claim 1, wherein the synthetic DNA isselected from one or more of: chemically synthesized DNA, PCR amplifiedDNA, engineered DNA and polynucleotides comprising nucleoside analogs.3. The scPV of claim 1, wherein the synthetic DNA is chemicallysynthesized DNA.
 4. The scPV of any one of claims 1 to 3, wherein theone or more modifications comprise one or more deletions, insertions,substitutions, or a combination thereof.
 5. (canceled)
 6. The scPV ofany one of claims 1 to 4, wherein the viral genome comprisesheterologous terminal hairpin loops.
 7. The scPV of any one of claims 1to 4 or claim 6, wherein the viral genome comprises terminal hairpinloops derived from vaccinia virus.
 8. The scPV of claim 7, wherein theleft and right terminal hairpin loops a) comprise the slow form and thefast form of the vaccinia virus terminal hairpin loop, respectively, b)comprise the fast form and the slow form of the vaccinia virus terminalhairpin loop, respectively, c) both comprise the slow form of thevaccinia virus terminal hairpin loop, or d) both comprise the fast formof the vaccinia virus terminal loop.
 9. The scPV of any one of claims 1to 4 or claims 6 to 8, wherein the virus is replicated and reactivatedfrom overlapping chemically synthesized DNA fragments that correspond tosubstantially all of the viral genome of the scPV.
 10. The scPV of claim9, wherein the virus is replicated and reactivated from 1-14 overlappingfragments. 11.-12. (canceled)
 13. The scPV of any one of claims 1 to 4or 6 to 10, wherein the virus is reactivated using leporipoxvirus-catalyzed recombination and reactivation.
 14. (canceled)
 15. Asynthetic chimeric orthopox virus (scOPV) that is replicated andreactivated from DNA derived from synthetic DNA, the viral genome ofsaid virus differing from a wild type genome of said virus in that it ischaracterized by one or more modifications, the modifications beingderived from a group comprising chemically synthesized DNA, cDNA orgenomic DNA.
 16. The scOPV of claim 15, wherein the synthetic DNA isselected from one or more of: chemically synthesized DNA, PCR amplifiedDNA, engineered DNA and polynucleotides comprising nucleoside analogs.17. The scOPV of claim 15, wherein the synthetic DNA is chemicallysynthesized DNA.
 18. The scOPV of any one of claims 15 to 17, whereinthe viral genome of the scOPV is based on an OPV selected from the groupconsisting of: camelpox (CMLV) virus, cowpox virus (CPXV), ectromeliavirus (ECTV), horsepox virus (HPXV), monkeypox virus (MPXV), vacciniavirus (VACV), variola virus (VARV), rabbitpox virus (RPXV), raccoonpoxvirus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus,and volepox virus.
 19. The scOPV of any one of claims 15 to 18, whereinthe viral genome of the scOPV is based on a VACV.
 20. The scOPV of claim19, wherein the viral genome of the scOPV is based on the genome of VACVstrain ACAM2000 and differs from the ACAM2000 genome in that it ischaracterized by one or more modifications.
 21. The scOPV of claim 19,wherein the viral genome of the scOPV is based on the genome of VACVstrain IOC and differs from the IOC genome in that it is characterizedby one or more modifications.
 22. The scOPV of claim 19, wherein theviral genome of the scOPV is based on the genome of VACV strain MVA anddiffers from the MVA genome in that it is characterized by one or moremodifications.
 23. (canceled)
 24. The scOPV of claim 19, wherein thewild type VACV genome is the genome of a strain selected from the groupconsisting of: Western Reserve, Clone 3, Tian Tian, Tian Tian clone TT9,Tian Tian clone TP3, NYCBH, Wyeth, Copenhagen, Lister 107, Lister-LO,IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR,Evans, Praha, LIVP, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, ConnaughtLaboratories, Serro 2, CM-01, Dryvax clone DPP13, Dryvax clone DPP15,Dryvax clone DPP20, Dryvax clone DPP17, Dryvax clone DPP21, andchorioallantois vaccinia virus Ankara.
 25. The scOPV of any one ofclaims 15 to 22 or 24, wherein the one or more modifications compriseone or more deletions, insertions, substitutions, or a combinationthereof. 26.-30. (canceled)
 31. The scOPV of any one of claims 15 to 22or 24 or 25, wherein the viral genome comprises heterologous terminalhairpin loops.
 32. The scOPV of any one of claims 15 to 22 or 24 or 25,wherein the viral genome comprises terminal hairpin loops derived fromvaccinia virus.
 33. The scOPV of claim 32, wherein the left and rightterminal hairpin loops a) comprise the slow form and the fast form ofthe vaccinia virus terminal hairpin loop, respectively, b) comprise thefast form and the slow form of the vaccinia virus terminal hairpin loop,respectively, c) both comprise the slow form of the vaccinia virusterminal hairpin loop, or d) both comprise the fast form of the vacciniavirus terminal loop.
 34. The scOPV of claim 33, wherein the slow formcomprises a nucleotide sequence that is at least 85% identical to thenucleotide sequence of SEQ ID NO: 11 and the fast form comprises anucleotide sequence that is at least 85% identical to the nucleotidesequence of SEQ ID NO:
 12. 35.-36. (canceled)
 37. The scOPV of claim 33,wherein the slow form consists of the nucleotide sequence of SEQ ID NO:11 and the fast form consists of the nucleotide sequence of SEQ ID NO:12.
 38. The scOPV of any one of claims 15 to 22 or 24 or 25 or 31 to 34or 37, wherein the virus is replicated and reactivated from overlappingchemically synthesized DNA fragments that correspond to substantiallyall of the viral genome of the OPV.
 39. The scOPV of claim 38, whereinthe virus is replicated and reactivated from 1-14 overlapping fragments.40.-41. (canceled)
 42. The scOPV of any one of claims 15 to 22 or 24 or25 or 31 to 34 or 37 to 39, wherein the virus is reactivated usingleporipox virus-catalyzed recombination and reactivation.
 43. (canceled)44. A synthetic chimeric horsepox virus (scHPXV) that is replicated andreactivated from synthetic DNA, the viral genome differing from a wildtype genome of HPXV in that it is characterized by one or moremodifications, the modifications being derived from a group comprisingchemically synthesized DNA, cDNA or genomic DNA.
 45. The scHPXV of claim44, wherein the synthetic DNA is selected from one or more of:chemically synthesized DNA, PCR amplified DNA, engineered DNA andpolynucleotides comprising nucleoside analogs.
 46. The scHPXV of claim44, wherein the synthetic DNA is chemically synthesized DNA.
 47. ThescHPXV of any one of claims 44 to 46, wherein the viral genome is basedon the genome of HPXV strain MNR-76 and differs from the MNR-76 genomein that it is characterized by one or more modifications.
 48. The scHPXVof any one of claims 44 to 47, wherein the one or more modificationscomprise one or more deletions, insertions, substitutions, or acombination thereof. 49.-55. (canceled)
 56. The scHPXV of any one ofclaims 44 to 48, wherein the one or more modifications comprise one ormore mutations listed in Table
 2. 57. The scHPXV of any one of claims 44to 48 or 56, wherein the viral genome comprises heterologous terminalhairpin loops.
 58. The scHPXV of claim 57, wherein the viral genomecomprises terminal hairpin loops derived from vaccinia virus.
 59. ThescHPXV of claim 58, wherein the left and right terminal hairpin loops a)comprise the slow form and the fast form of the vaccinia virus terminalhairpin loop, respectively, b) comprise the fast form and the slow formof the vaccinia virus terminal hairpin loop, respectively, c) bothcomprise the slow form of the vaccinia virus terminal hairpin loop, ord) both comprise the fast form of the vaccinia virus terminal loop. 60.The scHPXV of claim 59, wherein the slow form comprises a nucleotidesequence that is at least 85% identical to the sequence of SEQ ID NO: 11and the fast form comprises a nucleotide sequence that is at least 85%identical to the nucleotide sequence of SEQ ID NO:
 12. 61.-62.(canceled)
 63. The scHPXV of claim 59, wherein the slow form consists ofthe nucleotide sequence of SEQ ID NO: 11 and the fast form consists ofthe nucleotide sequence of SEQ ID NO:
 12. 64. The scHPXV of claim 57,wherein the viral genome comprises terminal hairpin loops derived fromcamelpox virus, cowpox virus, ectromelia virus, monkeypox virus, variolavirus, rabbitpox virus, raccoon poxvirus, skunkpox virus, Taterapoxvirus, Uasin Gishu disease virus, or volepox virus.
 65. The scHPXV ofany one of claims 44 to 48 or 56 to 60 or 63 or 64, wherein the virus isreplicated and assembled from overlapping chemically synthesized DNAfragments that correspond to substantially all of the viral genome ofHPXV.
 66. The scHPXV of claim 65, wherein the virus is replicated andreactivated from 1-14 overlapping fragments. 67.-68. (canceled)
 69. ThescHPXV of any one of claims 44 to 48 or 56 to 60 or 63 to 66, whereinthe virus is reactivated using leporipox virus-catalyzed recombinationand reactivation.
 70. (canceled)
 71. A method of producing a syntheticchimeric poxvirus (scPV) comprising the steps of: (i) chemicallysynthesizing overlapping DNA fragments that correspond to substantiallyall of the viral genome of the poxvirus; (ii) transfecting theoverlapping DNA fragments into helper virus-infected cells; (iii)culturing said cells to produce a mixture of helper virus and syntheticchimeric poxviral particles in said cells; and (iv) plating the mixtureon host cells specific to the scPV to recover the scPV.
 72. The methodof claim 71, wherein the helper virus is a leporipox virus. 73.-74.(canceled)
 75. The method of claim 71, wherein the helper virus isfowlpox virus.
 76. The method of claim 71, wherein the helper virus is apsoralen-inactivated helper virus.
 77. (canceled)
 78. The method of anyone of claims 71 to 72, wherein step (i) further comprises chemicallysynthesizing terminal hairpin loops from a poxvirus and ligating themonto the fragments comprising the left and right termini of the viralgenome.
 79. A method of producing a synthetic chimeric orthopox virus(scOPV) comprising the steps of: (i) chemically synthesizing overlappingDNA fragments that correspond to substantially all of the viral genomeof the OPV; (ii) transfecting the overlapping DNA fragments into helpervirus-infected cells; (iii) culturing said cells to produce a mixture ofhelper virus and scOPV particles in said cells; and (iv) plating themixture on OPV-specific host cells to recover the scOPV.
 80. The methodof claim 79, wherein the helper virus is a leporipox virus. 81.-82.(canceled)
 83. The method of claim 79, wherein the helper virus isfowlpox virus.
 84. The method of claim 79, wherein the helper virus is apsoralen-inactivated helper virus. 85.-86. (canceled)
 87. The method ofany one of claims 79 or 80 or 83 or 84, wherein the OPV is selected fromthe group consisting of: camelpox virus, cowpox virus, ectromelia virus,horsepox virus, monkeypox virus, vaccinia virus, variola virus,rabbitpox virus, raccoon poxvirus, skunkpox virus, Taterapox virus,Uasin Gishu disease virus, volepox virus.
 88. The method of any one ofclaims 79 or 80 or 83 or 84 or 87, wherein step (i) further compriseschemically synthesizing terminal hairpin loops from an OPV and ligatingthem onto the fragments comprising the left and right termini of theviral genome.
 89. A method of producing a synthetic chimeric horsepoxvirus (scHPXV) comprising the steps of: (i) chemically synthesizingoverlapping DNA fragments that correspond to substantially all of theHPXV genome; (ii) transfecting the overlapping DNA fragments into helpervirus-infected cells; (iii) culturing said cells to produce a mixture ofhelper virus and scHPXV particles in said cells; and (iv) plating themixture on HPXV-specific host cells to recover the scHPXV.
 90. Themethod of claim 89, wherein the helper virus is a leporipox virus.91.-92. (canceled)
 93. The method of claim 89, wherein the helper virusis fowlpox virus.
 94. The method of claim 89, wherein the helper virusis a psoralen-inactivated helper virus. 95.-96. (canceled)
 97. Themethod of any one of claims 89 or 90 or 93 or 94, wherein step (i)further comprises chemically synthesizing terminal hairpin loops from anOPV and ligating them onto the fragments comprising the left and righttermini of the HPXV genome.
 98. The method of any one of claims 89 or 90or 93 or 94 or 97, wherein the overlapping DNA fragments comprise: i)nucleotide sequences that are at least 85% identical to the sequences ofSEQ ID NOs: 1-10; ii) nucleotide sequences that are at least 90%identical to the sequences of SEQ ID NOs: 1-10; (iii) nucleotidesequences that are at least 95% identical to the sequences of SEQ IDNOs: 1-10; or (iv) nucleotide sequences that consist of the sequences ofSEQ ID NOs: 1-10.
 99. A method of producing a synthetic chimerichorsepox virus (scHPXV) comprising: (i) chemically synthesizingoverlapping DNA fragments that correspond to substantially all of theHPXV genome; (ii) transfecting the overlapping DNA fragments into Shopefibroma virus (SFV)-infected cells; (iii) culturing said cells toproduce a mixture of SFV and scHPXV particles in said cells; and (iv)plating the mixture on HPXV-specific host cells to recover the scHPXV.100.-101. (canceled)
 102. A synthetic chimeric poxvirus (scPV) generatedby the method of any one of claims 71 to 72 or 75 or 76 or
 78. 103. Asynthetic chimeric orthopox virus (scOPV) generated by the method of anyone of claims 79 or 80 or 83 or 84 or 87 or
 88. 104. A syntheticchimeric horsepox virus (scHPXV) generated by the method of any one ofclaims 89 or 90 or 93 or 94 or 97 to
 99. 105. A composition comprising apharmaceutically acceptable carrier and the scPV of any one of claims 1to 4 or 6 to 10 or 13 or
 102. 106. A composition comprising apharmaceutically acceptable carrier and the scOPV of any one of claims15 to 22 or 24 or 25 or 31 to 34 or 37 to 39 or 42 or
 103. 107. A methodof triggering or boosting an immune response against variola virus,comprising administering to a subject in need thereof a compositioncomprising the scOPV of any one of claims 15 to 22 or 24 or 25 or 31 to34 or 37 to 39 or 42 or
 103. 108. A method of triggering or boosting animmune response against vaccinia virus, comprising administering to asubject in need thereof a composition comprising the scOPV of any one ofclaims 15 to 22 or 24 or 25 or 31 to 34 or 37 to 39 or 42 or
 103. 109. Amethod of triggering or boosting an immune response against monkeypoxvirus, comprising administering to a subject in need thereof acomposition comprising the scOPV of any one of claims 15 to 22 or 24 or25 or 31 to 34 or 37 to 39 or 42 or
 103. 110. A method of immunizing ahuman subject to protect said subject from variola virus infection,comprising administering to said subject a composition comprising thescOPV of any one of claims 15 to 22 or 24 or 25 or 31 to 34 or 37 to 39or 42 or
 103. 111. A method of treating a variola virus infection,comprising administering to a subject in need thereof a compositioncomprising the scOPV of any one of claims 15 to 22 or 24 or 25 or 31 to34 or 37 to 39 or 42 or
 103. 112. A composition comprising apharmaceutically acceptable carrier and the scHPXV of any one of claims44 to 48 or 56 to 60 or 63 to 66 or 69 or
 104. 113. A method oftriggering or boosting an immune response against variola virus,comprising administering to a subject in need thereof a compositioncomprising the scHPXV of any one of claims 44 to 48 or 56 to 60 or 63 to66 or 69 or
 104. 114. A method of triggering or boosting an immuneresponse against vaccinia virus, comprising administering to a subjectin need thereof a composition comprising the scHPXV of any one of claims44 to 48 or 56 to 60 or 63 to 66 or 69 or
 104. 115. A method oftriggering or boosting an immune response against monkeypox virus,comprising administering to a subject in need thereof a compositioncomprising the scHPXV of any one of claims 44 to 48 or 56 to 60 or 63 to66 or 69 or
 104. 116. A method of immunizing a human subject to protectsaid subject from variola virus infection, comprising administering tosaid subject a composition comprising the scHPXV of any one of claims 44to 48 or 56 to 60 or 63 to 66 or 69 or
 104. 117. A method of treating avariola virus infection, comprising administering to a subject in needthereof a composition comprising the scHPXV of any one of claims 44 to48 or 56 to 60 or 63 to 66 or 69 or
 104. 118.-123. (canceled)
 124. Apoxvirus treatment facility wherein subjects in need of immunization ortreatment with a composition comprising the virus of any one of claims 1to 4 or 6 to 10 or 13 or 15 to 22 or 24 or 25 or 31 to 34 or 37 to 39 or42 or 44 to 48 or 56 to 60 or 63 to 66 or 69 or 102 to 106 or thecomposition of any one of claim 105, 106, or 112 may be immunized ortreated in an environment such that they are sequestered from othersubjects not intended to be immunized or treated.
 125. The poxvirustreatment facility of claim 124 being characterized by specialists insmallpox adverse events to administer the immunization or treatment.126. (canceled)
 127. The poxvirus treatment facility of any one of claim124 or 125, wherein the subjects in need of immunization or treatmentare immunized or treated according to the methods of any one of claims107 to 111 or 113 to 117.